https://www.biotechnologyforums.com/ Wed, 22 Apr 2026 14:09:43 +0000 MyBB https://www.biotechnologyforums.com/thread-7814.html Wed, 22 Feb 2017 12:11:56 +0000 CHANDA7]]> https://www.biotechnologyforums.com/thread-7814.html If we, please help me with the answer.....

THANKS.....]]> If we, please help me with the answer.....

THANKS.....]]> https://www.biotechnologyforums.com/thread-7317.html Mon, 09 May 2016 14:16:34 +0000 gene26]]> https://www.biotechnologyforums.com/thread-7317.html Has anyone ever tried to produce MAb with a peptide (20 a.a sequence) with FCA adjuvant alone, without any carrier protein. Does it work, If so could you direct me to a paper?

the other group tried KLH conjugate. I'am trying without carrier protein with adjuvant, I don't see nice titer values. Is this even a feasible approach. 


people had a problem with carrier that antibodies were raised against the carrier and very low against desired epitope. Is there any paper that worked out peptide without carrier. I see in every book they mention associating with carrier protein .]]> Has anyone ever tried to produce MAb with a peptide (20 a.a sequence) with FCA adjuvant alone, without any carrier protein. Does it work, If so could you direct me to a paper?

the other group tried KLH conjugate. I'am trying without carrier protein with adjuvant, I don't see nice titer values. Is this even a feasible approach. 


people had a problem with carrier that antibodies were raised against the carrier and very low against desired epitope. Is there any paper that worked out peptide without carrier. I see in every book they mention associating with carrier protein .]]> https://www.biotechnologyforums.com/thread-2360.html Mon, 15 Jul 2013 19:06:33 +0000 rasing02]]> https://www.biotechnologyforums.com/thread-2360.html
If have any information on the same then please share and I would also like information on how to obtain these vectors. I would ideally like to have a single vector in which multiple genes can be cloned and expressed in plants. If not then I would like to have some more information on what criteria I should use in selecting two different vectors and choose which plant for transgenic expression.

I hope I have explained myself well. If you have any questions please ask me.
Thanks!]]>
If have any information on the same then please share and I would also like information on how to obtain these vectors. I would ideally like to have a single vector in which multiple genes can be cloned and expressed in plants. If not then I would like to have some more information on what criteria I should use in selecting two different vectors and choose which plant for transgenic expression.

I hope I have explained myself well. If you have any questions please ask me.
Thanks!]]> https://www.biotechnologyforums.com/thread-2189.html Sat, 04 May 2013 09:04:57 +0000 NoahMachuki]]> https://www.biotechnologyforums.com/thread-2189.html Two technologies possible for human cloning include Artificial embryo twinning and Somatic Cell Nuclear Transfer.

Cloning is the developing of an organism that has a complete genetic makeup of another. The two organisms, the clone and the original organism could be explained better by a scenario of identical twins. Identical twins have similar composition of all their genes therefore, giving them the characteristic of similarity. Over years, clones of different organisms like mice, frogs and pets have been developed. Actually, cloning is dated back to early 1970’s. The cloning of these organisms some of which include mammals has given insight to a notable ability to develop human clones. Actually the prospect of human cloning first came to lame light after the successful cloning of Dolly the lamb in 1997. The implementation of human cloning has not been done due to imbalance in its pros and cons. Nevertheless, clones of numerous other organisms have been developed.

Many clones and the technology itself have proved beneficial and have been accepted in the society. Clones have provided disease models. When infected with disease causing agents they are efficiently used in acquiring insight of the disease. Clones of stem cells have been sufficiently used in repairing of tissues that are damaged. Cloning again has enabled to generate great numbers of transgenic organisms. These are organisms that produce commercially viable products such as proteins. Another applicable advantage of cloning would be in reviving extinct organisms and sustaining species that face danger of extinction. However, reviving the extinct organisms would rather be theoretical than practical, unless undamaged and properly kept DNA samples of the organism are available.

Two technologies possible for human cloning include artificial embryo twinning and Somatic Cell Nuclear Transfer. Artificial embryo twinning technique is similar to the natural procedure of formation of identical twins. Naturally, a zygote divides into two or more cells in the same embryo. Each cell then develops resulting to identical individuals since they are genetically similar. The major difference between artificial embryo twinning and natural procedure lies in the environment used in developing the embryo. The latter occurs in a mother’s body while artificial embryo twinning is done in a Petri dish. Twinning is then done to the embryo resulting to two cells which are then allowed to grow individually in surrogate mother. The developed individuals are genetically identical since they originate from the same zygote.

Somatic cell nuclear transfer yields similar results to artificial embryo twinning although it makes use of ultimately different procedure. Somatic cells are all cells apart from the reproductive cells, the egg and sperm. The nucleus of a somatic cell is isolated and transferred to an egg cell whose nucleus is also removed prior. The egg cell, with its new nucleus develops into an embryo which is placed in surrogate mother for development. The resulting individual or the clone is genetically similar to the nucleus donor. However, it should be noted that the number of chromosomes inherited by the clones in both technologies is different. In artificial embryo twinning, both the sperm and egg donate a chromosome each. The embryo inherits two chromosomes and therefore, the clone ends up with two chromosomes. In somatic nuclear cell transfer, the nucleus of the recipient egg is removed prior to introduction of a new nucleus. Therefore the clone bears only a single chromosome.

These procedures give insight in the ability and possibility of developing human clones. However, the ultimate decision to clone human depends on the pros and cons of the same, which are discussed below. Human cloning would greatly help in medical research by providing absolutely actual environments to study certain medical conditions that have been of concern over years such as, growth of tumors and cancer. The study of cells would again give an insight to human aging. The study of human genetics would be easier and probably more effective, given that models similar to their counterparts are used. Considered a great benefit of this technology would be the ability to replace deceased underage humans, especially children. Children who die in accidents and acute diseases could be replaced. Using the somatic cell nuclear transfer method, nucleus of a somatic cell of the deceased could be isolated and used in developing a clone. Equally, celebrities and people who made great contributions in this world could be sustained by developing clones and therefore, help sustain talents and man power. Infertile couples would be able to bear children of their own kind through Somatic cell nuclear transfer. Again the bearing of twins would be out of human will other than nature. From an unbiased point of view, these benefits outwit the disadvantages of human cloning.

Challenges related to human cloning include; high chances of failure, problems during a later stage in development and abnormal expression of genes. These could probably be fixed as practice goes on. The chances of failure could be attributed to; first, incompatibility between a nucleus and the host egg cell. Secondly, it is not guaranteed that an egg induced with a foreign nucleus could definitely divide. Thirdly, failure could occur in introducing an embryo to the surrogate mother and/or further development in the surrogate mother could fail. Assuming that a certain clone is delivered, its normality cannot be predicted. Most clones suffer from “Large Offspring Syndrome’’. This is a condition whereby, a clone at birth will have larger organs than their natural counterparts. Organ malformations are also rampant hence severe complications later in life. Another challenge is that a clone could fail to express a required gene at the right place and in the right time. Failure to express the right gene at the right place and time could cause complications such as untimely aging and death. Therefore, there is never any estimated lifespan of a clone.

Prospects in human cloning face several challenges which include; legal, social and ethical issues. However, any technology that relieves human pain and trauma would be much welcome to the society. As the technology become successful, human would probably drop criticism and embrace it. It is recommended that, before arguing against human cloning and terming it as impunity, the positive impacts of this technology should be considered.]]> Two technologies possible for human cloning include Artificial embryo twinning and Somatic Cell Nuclear Transfer.

Cloning is the developing of an organism that has a complete genetic makeup of another. The two organisms, the clone and the original organism could be explained better by a scenario of identical twins. Identical twins have similar composition of all their genes therefore, giving them the characteristic of similarity. Over years, clones of different organisms like mice, frogs and pets have been developed. Actually, cloning is dated back to early 1970’s. The cloning of these organisms some of which include mammals has given insight to a notable ability to develop human clones. Actually the prospect of human cloning first came to lame light after the successful cloning of Dolly the lamb in 1997. The implementation of human cloning has not been done due to imbalance in its pros and cons. Nevertheless, clones of numerous other organisms have been developed.

Many clones and the technology itself have proved beneficial and have been accepted in the society. Clones have provided disease models. When infected with disease causing agents they are efficiently used in acquiring insight of the disease. Clones of stem cells have been sufficiently used in repairing of tissues that are damaged. Cloning again has enabled to generate great numbers of transgenic organisms. These are organisms that produce commercially viable products such as proteins. Another applicable advantage of cloning would be in reviving extinct organisms and sustaining species that face danger of extinction. However, reviving the extinct organisms would rather be theoretical than practical, unless undamaged and properly kept DNA samples of the organism are available.

Two technologies possible for human cloning include artificial embryo twinning and Somatic Cell Nuclear Transfer. Artificial embryo twinning technique is similar to the natural procedure of formation of identical twins. Naturally, a zygote divides into two or more cells in the same embryo. Each cell then develops resulting to identical individuals since they are genetically similar. The major difference between artificial embryo twinning and natural procedure lies in the environment used in developing the embryo. The latter occurs in a mother’s body while artificial embryo twinning is done in a Petri dish. Twinning is then done to the embryo resulting to two cells which are then allowed to grow individually in surrogate mother. The developed individuals are genetically identical since they originate from the same zygote.

Somatic cell nuclear transfer yields similar results to artificial embryo twinning although it makes use of ultimately different procedure. Somatic cells are all cells apart from the reproductive cells, the egg and sperm. The nucleus of a somatic cell is isolated and transferred to an egg cell whose nucleus is also removed prior. The egg cell, with its new nucleus develops into an embryo which is placed in surrogate mother for development. The resulting individual or the clone is genetically similar to the nucleus donor. However, it should be noted that the number of chromosomes inherited by the clones in both technologies is different. In artificial embryo twinning, both the sperm and egg donate a chromosome each. The embryo inherits two chromosomes and therefore, the clone ends up with two chromosomes. In somatic nuclear cell transfer, the nucleus of the recipient egg is removed prior to introduction of a new nucleus. Therefore the clone bears only a single chromosome.

These procedures give insight in the ability and possibility of developing human clones. However, the ultimate decision to clone human depends on the pros and cons of the same, which are discussed below. Human cloning would greatly help in medical research by providing absolutely actual environments to study certain medical conditions that have been of concern over years such as, growth of tumors and cancer. The study of cells would again give an insight to human aging. The study of human genetics would be easier and probably more effective, given that models similar to their counterparts are used. Considered a great benefit of this technology would be the ability to replace deceased underage humans, especially children. Children who die in accidents and acute diseases could be replaced. Using the somatic cell nuclear transfer method, nucleus of a somatic cell of the deceased could be isolated and used in developing a clone. Equally, celebrities and people who made great contributions in this world could be sustained by developing clones and therefore, help sustain talents and man power. Infertile couples would be able to bear children of their own kind through Somatic cell nuclear transfer. Again the bearing of twins would be out of human will other than nature. From an unbiased point of view, these benefits outwit the disadvantages of human cloning.

Challenges related to human cloning include; high chances of failure, problems during a later stage in development and abnormal expression of genes. These could probably be fixed as practice goes on. The chances of failure could be attributed to; first, incompatibility between a nucleus and the host egg cell. Secondly, it is not guaranteed that an egg induced with a foreign nucleus could definitely divide. Thirdly, failure could occur in introducing an embryo to the surrogate mother and/or further development in the surrogate mother could fail. Assuming that a certain clone is delivered, its normality cannot be predicted. Most clones suffer from “Large Offspring Syndrome’’. This is a condition whereby, a clone at birth will have larger organs than their natural counterparts. Organ malformations are also rampant hence severe complications later in life. Another challenge is that a clone could fail to express a required gene at the right place and in the right time. Failure to express the right gene at the right place and time could cause complications such as untimely aging and death. Therefore, there is never any estimated lifespan of a clone.

Prospects in human cloning face several challenges which include; legal, social and ethical issues. However, any technology that relieves human pain and trauma would be much welcome to the society. As the technology become successful, human would probably drop criticism and embrace it. It is recommended that, before arguing against human cloning and terming it as impunity, the positive impacts of this technology should be considered.]]> https://www.biotechnologyforums.com/thread-1950.html Sat, 29 Dec 2012 09:18:07 +0000 rhnegnet]]> https://www.biotechnologyforums.com/thread-1950.html I run a community of rh negative people and the claim that rh negative blood cannot be cloned keeps popping up. I would like to know from some of you if
1) this is correct
and
2) why.

Best,

Mike Dammann]]> I run a community of rh negative people and the claim that rh negative blood cannot be cloned keeps popping up. I would like to know from some of you if
1) this is correct
and
2) why.

Best,

Mike Dammann]]> https://www.biotechnologyforums.com/thread-1870.html Mon, 19 Nov 2012 12:29:02 +0000 Blackmoa]]> https://www.biotechnologyforums.com/thread-1870.html
Is it possible for sepal leaves to get chromosome number n, or are they always 2n?]]>
Is it possible for sepal leaves to get chromosome number n, or are they always 2n?]]> https://www.biotechnologyforums.com/thread-1856.html Fri, 16 Nov 2012 07:25:29 +0000 ashwathi]]> https://www.biotechnologyforums.com/thread-1856.html Transgenic Animals Detection:

In recombinant DNA technology after the gene of interest has been isolated and transferred efficiently, the next major step is to ensure its proper and maximum expression in order to obtain proposed results. Thus in a group of transgenic animals it is important that the animal with proper transgenic integration is identified.
Identification of transgenic animals:
The identification of transgenic animals is done using different mechanism.

In animals, where the transgene produces a distinctive phenotypic effect, the transgenic successful animals can be noted easily. But the results of all the gene transfer does not result in such distinct effects therefore other techniques have to be employed. The dot blot technique and PCR technique are to name a few.

Dots blot technique:
This technique helps in detecting several samples in one experiment. The sample of DNA collected is fixed onto a support like nitro cellulose filter. This then undergoes denaturation so that the double helix separates. Such membrane containing denatured sample when treated with radioactively labelled probe of corresponding transgene, the sample DNA incorporated with transgene binds with the probe. Upon removal of free probes by washing and analysed by autoradiography, it reveals presence of transgene which can be detected by fluorescence produced by radioactive probes. The strength of radioactivity exhibited shows the strength of transgene integration.

PCR technique:
This is the most important and wide speed used technology to identify the transgenic successful animals. The primers corresponding to the integrated transgene is used to amplify the test DNA sequences isolated from the transgenic animals. This results in the amplification of the transgene. The amplified DNA when blotted and hybridized, the presence of transgene is confirmed. But the techniques adopted for identification of transgenic animals does not reveal the actual level and site of integration. For achieving these further steps has to be adopted like:

Analysis of transgene integration:
In order to analyse the transgene integrated, the isolated gene samples which are detected with transgene integration, undergo restriction digestion with known restriction endonucleases. The fragments are separated by agarose gel electrophoresis and these fragments are analysed by southern blotting procedure. The fragments on the gel are transferred to a nitrocellulose filter membrane and denatured and probed with radioactive probe to reveal the actual site of transgenic integration. The integration of transgene can be confirmed by choosing the appropriate restriction enzyme. The presence of single or multiple integration of transgene is indicated by southern blotting.

Analysis of mRNA production:
The mRNA produced from a transgene can be detected as it is different from the native mRNA of the organisms. These can be purified and hybridized with a radioactive probe to detect mRNA production.

Analysis of protein expression:
The final aim of transgenic integration is to produce proteins coded by the transgene and its efficient expression. Therefore the detection of transgene integration is possible by protein expression of the transgene. The analysis of transgenic proteins can be done by two methods: by identifying specific antibodies produced by transgenic proteins and by studying the enzymatic properties of the concerned proteins. In the case of antibody identification, various immunologic assays can be used. ELISA test is a classic example of the same. In this case, the antibody specific to the antigenic protein is added and allowed to react. Following the first reaction, this is further reacted with an antibody specific to the former antibody results in formation of a complex. This when treated with the substrate of corresponding enzyme, it produces colour proportional to the strength of the antibody indicating the amount of corresponding transgenic protein expressed. The other assays included are immunoblotting, radioimmunoprecipitation, immunohistochemical staining etc.

Enzyme activities can be detected by using transgenic genes which produces different enzymatic activities or different pathway which is not shown by host enzymes. Various procedures can be employed to detect this. The main procedure followed is by transfer of scorable marker genes. Scorable marker genes are those which produce a definite phenotype. Main examples for these are chloramphenicol acetyltransferase (CAT) genes used mainly in fish or mammal cells, betagalactocidase and luciferase gene used in fish.

All this analysis has to be repeated in two or more different potentially transgenic animals so as to determine the level of transgenic integration as, in different organisms it tend to differ. This occurs because the site of integration may differ among animals. The animals has to be studied to confirm which site integration yield maximum expression and further research must be forwarded to achieve the same.]]> Transgenic Animals Detection:

In recombinant DNA technology after the gene of interest has been isolated and transferred efficiently, the next major step is to ensure its proper and maximum expression in order to obtain proposed results. Thus in a group of transgenic animals it is important that the animal with proper transgenic integration is identified.
Identification of transgenic animals:
The identification of transgenic animals is done using different mechanism.

In animals, where the transgene produces a distinctive phenotypic effect, the transgenic successful animals can be noted easily. But the results of all the gene transfer does not result in such distinct effects therefore other techniques have to be employed. The dot blot technique and PCR technique are to name a few.

Dots blot technique:
This technique helps in detecting several samples in one experiment. The sample of DNA collected is fixed onto a support like nitro cellulose filter. This then undergoes denaturation so that the double helix separates. Such membrane containing denatured sample when treated with radioactively labelled probe of corresponding transgene, the sample DNA incorporated with transgene binds with the probe. Upon removal of free probes by washing and analysed by autoradiography, it reveals presence of transgene which can be detected by fluorescence produced by radioactive probes. The strength of radioactivity exhibited shows the strength of transgene integration.

PCR technique:
This is the most important and wide speed used technology to identify the transgenic successful animals. The primers corresponding to the integrated transgene is used to amplify the test DNA sequences isolated from the transgenic animals. This results in the amplification of the transgene. The amplified DNA when blotted and hybridized, the presence of transgene is confirmed. But the techniques adopted for identification of transgenic animals does not reveal the actual level and site of integration. For achieving these further steps has to be adopted like:

Analysis of transgene integration:
In order to analyse the transgene integrated, the isolated gene samples which are detected with transgene integration, undergo restriction digestion with known restriction endonucleases. The fragments are separated by agarose gel electrophoresis and these fragments are analysed by southern blotting procedure. The fragments on the gel are transferred to a nitrocellulose filter membrane and denatured and probed with radioactive probe to reveal the actual site of transgenic integration. The integration of transgene can be confirmed by choosing the appropriate restriction enzyme. The presence of single or multiple integration of transgene is indicated by southern blotting.

Analysis of mRNA production:
The mRNA produced from a transgene can be detected as it is different from the native mRNA of the organisms. These can be purified and hybridized with a radioactive probe to detect mRNA production.

Analysis of protein expression:
The final aim of transgenic integration is to produce proteins coded by the transgene and its efficient expression. Therefore the detection of transgene integration is possible by protein expression of the transgene. The analysis of transgenic proteins can be done by two methods: by identifying specific antibodies produced by transgenic proteins and by studying the enzymatic properties of the concerned proteins. In the case of antibody identification, various immunologic assays can be used. ELISA test is a classic example of the same. In this case, the antibody specific to the antigenic protein is added and allowed to react. Following the first reaction, this is further reacted with an antibody specific to the former antibody results in formation of a complex. This when treated with the substrate of corresponding enzyme, it produces colour proportional to the strength of the antibody indicating the amount of corresponding transgenic protein expressed. The other assays included are immunoblotting, radioimmunoprecipitation, immunohistochemical staining etc.

Enzyme activities can be detected by using transgenic genes which produces different enzymatic activities or different pathway which is not shown by host enzymes. Various procedures can be employed to detect this. The main procedure followed is by transfer of scorable marker genes. Scorable marker genes are those which produce a definite phenotype. Main examples for these are chloramphenicol acetyltransferase (CAT) genes used mainly in fish or mammal cells, betagalactocidase and luciferase gene used in fish.

All this analysis has to be repeated in two or more different potentially transgenic animals so as to determine the level of transgenic integration as, in different organisms it tend to differ. This occurs because the site of integration may differ among animals. The animals has to be studied to confirm which site integration yield maximum expression and further research must be forwarded to achieve the same.]]> https://www.biotechnologyforums.com/thread-1794.html Tue, 23 Oct 2012 07:13:44 +0000 ashwathi]]> https://www.biotechnologyforums.com/thread-1794.html Restriction endonuclease refers to a group of endonucleases which cleaves the DNA at specific points known as recognition sequences or sites. Different types of restriction endonucleases have been identified like type I, II, and III. Among these, the most available and most extensively used enzyme is type II restriction endonuclease. Examples are: ECoR I, Hind III, etc.

The importance of restriction enzymes lies in the property that it cleaves the DNA sequence, in most cases, within their specific recognition sequences unlike other restriction enzymes which cuts some base pairs away from their recognition sites. Some type II restriction endonucleases are also known to cleave the DNA sequence in close proximity of their recognition sequence rather than within the recognition site. This efficient nature of type II restriction endonucleases, combined with their comparatively smaller structure, has led to the wide application of these enzymes in gene cloning.

Mechanism of type II restriction endonucleases.
Recognition sites are specific area or sequences in the genetic molecule which these enzymes recognize as sites for cleavage. Recognition sites are unique for different restriction endonucleases. For type II restriction endonucleases, recognition sites are mostly palindromic sequences with rotational symmetry. DNA has a double stranded helical structure where, the nucleotides of the two strands of DNA are complementary to each other. There are certain sequences in such a structure where, the first half of the sequence is a mirror image of the second half of the complementary strand and reads identical from same end. Such sequences are termed as palindromic sequence with rotational symmetry.
Eg:-
5’GAATTC3’
3’CTTAAG5’

The restriction endonuclease moves along the surface of the DNA until it recognises its target sites. After recognition, it initiates DNA binding in the presence of Mg2+ ions resulting in cleavage at specific sites.

Cleavage:
The cleavage patterns produced by different restriction endonucleases are specific and each holds a novel role in gene cloning.
The two main patterns of cleavage are creating staggered cuts and even cuts. In staggered cuts, the cleavage occurs in different locations resulting in producing protruding ends of one of the strands in the double helix. Such ends are known as cohesive or sticky ends. The main benefit of such ends is that the protruding ends created are usually complementary in nature and can be used to link with vectors consisting complementary sequences for isolating the DNA fragment. It forms the basics for recombinant DNA techniques such as southern blotting. The even cuts, on the other hand, produces blunt ends where the two strands are cleaved at similar points. The importance of blunt ends in gene cloning involves many techniques which are utilized to modify the blunt ends in a manner so as to meet the specific requirements.

These include:
Tailing: This is a procedure which results in a protruding end of a defined length being created which aids in the pairing of required DNA segment with appropriate vector.

Linker: Linkers are chemically synthesized oligonucleotides. This can be used to modify the blunt ends so as to create cohesive ends of required bases. Linkers are so designed as to have a recognition site of a specific endonuclease. This can be linked to a blunt end DNA fragment created by the restriction digest. Such a modified fragment when digested by the linker specific restriction endonuclease can create cohesive ends complementary to vectors which can later be isolated to create multiple copies or, can be used in creating a recombinant DNA.

Adapters: These are short artificially synthesized double stranded fragments which can be used to link two blunt ends with different end sequence.

As a result of all these techniques, it is possible to alter a specific gene at a nucleotide level by identifying the respective restriction endonuclease enzyme which can cleave at the specific site. This is the principle adapted in gene cloning given that, to create a specific clone it is required to isolate the target gene. This isolation can only be done with the help of a restriction endonuclease enzyme. The type II restriction enzymes accentuates the importance as they have the ability to cleave at exact points resulting in producing definite fragments rather than random fragments.

Other Applications:
(i) RFLP (restricted fragment length polymorphism), which involves production of DNA fragments of different lengths which can be separated and utilized for several purposes like DNA fingerprinting, identification of mutations, preparation of genomic library etc.

(ii) A technique called restriction mapping make use of the capability of restriction enzymes to create DNA fragments of specific length thus distinguish alleles of a single gene having altered restriction sites.

(iii) Gene therapy: This employs the property of restriction endonuclease to recognize and remove a specific DNA fragment responsible for many diseases.]]> Restriction endonuclease refers to a group of endonucleases which cleaves the DNA at specific points known as recognition sequences or sites. Different types of restriction endonucleases have been identified like type I, II, and III. Among these, the most available and most extensively used enzyme is type II restriction endonuclease. Examples are: ECoR I, Hind III, etc.

The importance of restriction enzymes lies in the property that it cleaves the DNA sequence, in most cases, within their specific recognition sequences unlike other restriction enzymes which cuts some base pairs away from their recognition sites. Some type II restriction endonucleases are also known to cleave the DNA sequence in close proximity of their recognition sequence rather than within the recognition site. This efficient nature of type II restriction endonucleases, combined with their comparatively smaller structure, has led to the wide application of these enzymes in gene cloning.

Mechanism of type II restriction endonucleases.
Recognition sites are specific area or sequences in the genetic molecule which these enzymes recognize as sites for cleavage. Recognition sites are unique for different restriction endonucleases. For type II restriction endonucleases, recognition sites are mostly palindromic sequences with rotational symmetry. DNA has a double stranded helical structure where, the nucleotides of the two strands of DNA are complementary to each other. There are certain sequences in such a structure where, the first half of the sequence is a mirror image of the second half of the complementary strand and reads identical from same end. Such sequences are termed as palindromic sequence with rotational symmetry.
Eg:-
5’GAATTC3’
3’CTTAAG5’

The restriction endonuclease moves along the surface of the DNA until it recognises its target sites. After recognition, it initiates DNA binding in the presence of Mg2+ ions resulting in cleavage at specific sites.

Cleavage:
The cleavage patterns produced by different restriction endonucleases are specific and each holds a novel role in gene cloning.
The two main patterns of cleavage are creating staggered cuts and even cuts. In staggered cuts, the cleavage occurs in different locations resulting in producing protruding ends of one of the strands in the double helix. Such ends are known as cohesive or sticky ends. The main benefit of such ends is that the protruding ends created are usually complementary in nature and can be used to link with vectors consisting complementary sequences for isolating the DNA fragment. It forms the basics for recombinant DNA techniques such as southern blotting. The even cuts, on the other hand, produces blunt ends where the two strands are cleaved at similar points. The importance of blunt ends in gene cloning involves many techniques which are utilized to modify the blunt ends in a manner so as to meet the specific requirements.

These include:
Tailing: This is a procedure which results in a protruding end of a defined length being created which aids in the pairing of required DNA segment with appropriate vector.

Linker: Linkers are chemically synthesized oligonucleotides. This can be used to modify the blunt ends so as to create cohesive ends of required bases. Linkers are so designed as to have a recognition site of a specific endonuclease. This can be linked to a blunt end DNA fragment created by the restriction digest. Such a modified fragment when digested by the linker specific restriction endonuclease can create cohesive ends complementary to vectors which can later be isolated to create multiple copies or, can be used in creating a recombinant DNA.

Adapters: These are short artificially synthesized double stranded fragments which can be used to link two blunt ends with different end sequence.

As a result of all these techniques, it is possible to alter a specific gene at a nucleotide level by identifying the respective restriction endonuclease enzyme which can cleave at the specific site. This is the principle adapted in gene cloning given that, to create a specific clone it is required to isolate the target gene. This isolation can only be done with the help of a restriction endonuclease enzyme. The type II restriction enzymes accentuates the importance as they have the ability to cleave at exact points resulting in producing definite fragments rather than random fragments.

Other Applications:
(i) RFLP (restricted fragment length polymorphism), which involves production of DNA fragments of different lengths which can be separated and utilized for several purposes like DNA fingerprinting, identification of mutations, preparation of genomic library etc.

(ii) A technique called restriction mapping make use of the capability of restriction enzymes to create DNA fragments of specific length thus distinguish alleles of a single gene having altered restriction sites.

(iii) Gene therapy: This employs the property of restriction endonuclease to recognize and remove a specific DNA fragment responsible for many diseases.]]> https://www.biotechnologyforums.com/thread-1779.html Sat, 13 Oct 2012 17:16:13 +0000 BojanaL]]> https://www.biotechnologyforums.com/thread-1779.html
In 1972, Oliver Ryder, geneticist at San Diego Zoo decided to collect as much animal skin samples as possible, hoping that tissue bank might be useful in future attempts to save endangered animals. He didn’t know that animals will be created using somatic cells in the future. Stored tissues were named “Frozen Zoo” thanks to preservation method used (liquid nitrogen). Until now, this collection grew to impressive number of ~9 000 samples belonging to ~1 000 different vertebrate species. Although initial idea wasn’t to create a gene pool that will be used for animal cloning, latest techniques and high rate of extinction made “Frozen Zoo” serving that purpose exactly.

First attempts to clone threatened animal species started at the beginning of the 2001 using South Asian ox – guar. SCNT (stem cell nuclear transfer) using guar’s somatic cells and cow’s egg resulted in successful embryo development, that was seeded into cow’s uterus. Noah was born seemingly healthy, but 2 days later – he died due to infection.

In 2000, Spanish ibex, known as bucardo, went extinct. That was the first time that scientist tried to clone extinct animal. Nucleus derived from ibex’s skin cell was implanted into de-nucleated egg of domestic goat - its closest relative. New ibex was born, but died soon after the birth due to severe lung defect. As with Dolly the sheep, a lot of attempts were made before viable embryo that could survive until the birth was created. In the case of bucardo, 439 SCNT were made, 57 embryos were implanted, 7 embryos resulted in pregnancy and just one managed to survive until the birth. He lived for 7 minutes: inability to breath normally prevent him to live longer.

Success rate of cloning is 1%. Beside the low success rate, born animals are unhealthy and prone to various infections that doesn’t keep them alive for a long period.

In 2007, Japanese scientist concluded experiments on mice, revealing that adult somatic cells could be reverted to embryo like stem cells. Those cells were named “induced pluripotent stem cells” (iPS cells) and they could be used for creating any cell lineage you want. Oliver Ryder and his co-workers from San Diego Zoo are using iPS cells to create Northern white rhino, snow leopard and small West African monkey replicas as the number of those species is incredibly low (7 remaining individuals of white rhino are kept in captivity).
Besides “saving” animals that are still alive, or that are extinct few years ago, scientific appetites are growing bigger. How about creating Woolly Mammoth using DNA from his leftovers found under Siberian permafrost recently? Japanese and Russian scientist promise to create a mammoth in couple of years, using mammoth’s nucleus and elephant’s egg. Only problem with resurrecting mammoth is age of the DNA and damage found in his genetic material. However, using modern methods, 80% of mammoth genome is decoded. Creating chimera animal could solve the problem: stem cell derived from mammoth (using iPS method) placed near elephant embryo would affect early embryo development, resulting in animal having tissues created out of both mammoth and elephant cells. We are closer that we think to see mammoth (probably weak and unhealthy, but still successfully created) walking the Earth again.

What is making me really angry and sad is that mankind is responsible for all recently (couple of hundred years) noted extinctions. Why don't we use money and effort made in cloning animals to prevent extinction instead? Don’t you think that those kinds of experiments are cruel to the animals? Who ask female elephant if she is willing to be a surrogate mother to a mammoth? Would you like to be a surrogate mother to a chimpanzee, as its closest relative? I don’t think so. We managed to destroy so many beautiful things on the planet Earth, but driving animals extinct instead preserving them and then resurrecting them just to show future generation how mammoth looked like before he went extinct - I just don’t get it! If human ever become critically endangered – please don't clone me!]]>
In 1972, Oliver Ryder, geneticist at San Diego Zoo decided to collect as much animal skin samples as possible, hoping that tissue bank might be useful in future attempts to save endangered animals. He didn’t know that animals will be created using somatic cells in the future. Stored tissues were named “Frozen Zoo” thanks to preservation method used (liquid nitrogen). Until now, this collection grew to impressive number of ~9 000 samples belonging to ~1 000 different vertebrate species. Although initial idea wasn’t to create a gene pool that will be used for animal cloning, latest techniques and high rate of extinction made “Frozen Zoo” serving that purpose exactly.

First attempts to clone threatened animal species started at the beginning of the 2001 using South Asian ox – guar. SCNT (stem cell nuclear transfer) using guar’s somatic cells and cow’s egg resulted in successful embryo development, that was seeded into cow’s uterus. Noah was born seemingly healthy, but 2 days later – he died due to infection.

In 2000, Spanish ibex, known as bucardo, went extinct. That was the first time that scientist tried to clone extinct animal. Nucleus derived from ibex’s skin cell was implanted into de-nucleated egg of domestic goat - its closest relative. New ibex was born, but died soon after the birth due to severe lung defect. As with Dolly the sheep, a lot of attempts were made before viable embryo that could survive until the birth was created. In the case of bucardo, 439 SCNT were made, 57 embryos were implanted, 7 embryos resulted in pregnancy and just one managed to survive until the birth. He lived for 7 minutes: inability to breath normally prevent him to live longer.

Success rate of cloning is 1%. Beside the low success rate, born animals are unhealthy and prone to various infections that doesn’t keep them alive for a long period.

In 2007, Japanese scientist concluded experiments on mice, revealing that adult somatic cells could be reverted to embryo like stem cells. Those cells were named “induced pluripotent stem cells” (iPS cells) and they could be used for creating any cell lineage you want. Oliver Ryder and his co-workers from San Diego Zoo are using iPS cells to create Northern white rhino, snow leopard and small West African monkey replicas as the number of those species is incredibly low (7 remaining individuals of white rhino are kept in captivity).
Besides “saving” animals that are still alive, or that are extinct few years ago, scientific appetites are growing bigger. How about creating Woolly Mammoth using DNA from his leftovers found under Siberian permafrost recently? Japanese and Russian scientist promise to create a mammoth in couple of years, using mammoth’s nucleus and elephant’s egg. Only problem with resurrecting mammoth is age of the DNA and damage found in his genetic material. However, using modern methods, 80% of mammoth genome is decoded. Creating chimera animal could solve the problem: stem cell derived from mammoth (using iPS method) placed near elephant embryo would affect early embryo development, resulting in animal having tissues created out of both mammoth and elephant cells. We are closer that we think to see mammoth (probably weak and unhealthy, but still successfully created) walking the Earth again.

What is making me really angry and sad is that mankind is responsible for all recently (couple of hundred years) noted extinctions. Why don't we use money and effort made in cloning animals to prevent extinction instead? Don’t you think that those kinds of experiments are cruel to the animals? Who ask female elephant if she is willing to be a surrogate mother to a mammoth? Would you like to be a surrogate mother to a chimpanzee, as its closest relative? I don’t think so. We managed to destroy so many beautiful things on the planet Earth, but driving animals extinct instead preserving them and then resurrecting them just to show future generation how mammoth looked like before he went extinct - I just don’t get it! If human ever become critically endangered – please don't clone me!]]> https://www.biotechnologyforums.com/thread-1777.html Sat, 13 Oct 2012 10:48:03 +0000 BojanaL]]> https://www.biotechnologyforums.com/thread-1777.html
Molecular cloning is used to amplify DNA sequence (gene, promoter, non-coding sequence…) of interest. To ensure replication, DNA sequence must be linked with origin of replication – part of DNA that will initiate replication. Ligation is process of inserting DNA sequence into cloning vector (peace of DNA, carrier of the sequence). DNA ligaze will connect sequence and vector by “gluing” sticky ends at each DNA piece. Transfection of cell with vector carrying sequence of interest is next step. Electroporation, optical injection or biolistics are mostly used transfection techniques, but they are not successful always. Additional genes in cloning vector are necessary to ensure easy recognition of cells containing DNA piece of interest. Some of the most famous “markers” used are genes providing antibiotic resistance (when substrate with antibiotic is used for cell growing) or color markers (for blue/white cell screening). After cell colonies are formed, DNA sequence will be multiplied and analyzed using PCR, DNA sequencing or restriction fragment analysis.

Cellular cloning is production of cells containing same genetic info as mother cell. Cells derived from multi-cellular organism are much more complicated to clone than cells that are unicellular by nature. Technique “clone rings” is used for cloning multi-cellular organism derived cells. Cell suspension is exposed to mutagenic agent or drug and planted at high dilution, which result in new colonies formation. On a stage of few cells created, trypsin and polystyrene rings (covered in grease) are placed over each formed colony. Cells from the inner part of the ring are collected and moved to another substrate to develop further. Cellular cloning could solve serious medical issues that are non-treatable by conventional medication (such as Alzheimer disease). Cells used for this purpose are stem cells - as they could give raise to any cell lineage we want. SCNT (Stem Cell Nuclear Transport) is used for developing embryonic stem cells (ESC) that will have both research and therapeutic application. ESC are created by removing nucleus from the egg and implanting nucleus from adult somatic cell (containing both mother and father genetic material). Egg will act like it’s been fertilized and start dividing first to reach blastocyst stage and then toward any cell lineage we want. Procedure is the same with animal species and could be used to produce additional food source (by cloning farm animals) or to prevent extinction of endangered animals. It may sound like simple process, but success rate with this kind of genetic manipulation is pretty low. Dolly the sheep was first mammal created in laboratory. Out of 277 eggs used for SCNT, just 29 embryos were created. 3 survived until birth and only one - more famous by its given name – Dolly, survived until adulthood. Although, genetic material in the newly formed cell (organism) is the same as in donor’s cell, certain part of DNA is unique. Each cell contains mitochondria with its own genetic material. It’s inherited solely from the mother due to couple of reasons: egg contains more mtDNA than sperm; sperm derived mtDNA is easily degraded once inside or it can even fail to enter the egg. Thanks to this phenomenon, cloned cells can be considered genetic hybrids, as they contain both somatic DNA and mother mitochondrial genes.

Some large animals can create clones on their own. Lizards, snakes, ants, crustaceous species and even certain sharks are able to produce new individual by parthenogenesis – out of unfertilized egg. For most species, this is not obligatory way to reproduce but a method to overcome crisis in their environment. Komodo dragon, for example, can reproduce by parthenogenesis to increase the population in the habitat and then switch back to sexual reproduction to increase genetic diversity of the next generation.

To conclude, cloning is not something we invented, it’s natural phenomenon that we start exploiting recently. Wise and careful approach could be beneficial for the planet; we just need to pay attention not to cross the line, as genetic diversity is what allowed us to survive so far.]]>
Molecular cloning is used to amplify DNA sequence (gene, promoter, non-coding sequence…) of interest. To ensure replication, DNA sequence must be linked with origin of replication – part of DNA that will initiate replication. Ligation is process of inserting DNA sequence into cloning vector (peace of DNA, carrier of the sequence). DNA ligaze will connect sequence and vector by “gluing” sticky ends at each DNA piece. Transfection of cell with vector carrying sequence of interest is next step. Electroporation, optical injection or biolistics are mostly used transfection techniques, but they are not successful always. Additional genes in cloning vector are necessary to ensure easy recognition of cells containing DNA piece of interest. Some of the most famous “markers” used are genes providing antibiotic resistance (when substrate with antibiotic is used for cell growing) or color markers (for blue/white cell screening). After cell colonies are formed, DNA sequence will be multiplied and analyzed using PCR, DNA sequencing or restriction fragment analysis.

Cellular cloning is production of cells containing same genetic info as mother cell. Cells derived from multi-cellular organism are much more complicated to clone than cells that are unicellular by nature. Technique “clone rings” is used for cloning multi-cellular organism derived cells. Cell suspension is exposed to mutagenic agent or drug and planted at high dilution, which result in new colonies formation. On a stage of few cells created, trypsin and polystyrene rings (covered in grease) are placed over each formed colony. Cells from the inner part of the ring are collected and moved to another substrate to develop further. Cellular cloning could solve serious medical issues that are non-treatable by conventional medication (such as Alzheimer disease). Cells used for this purpose are stem cells - as they could give raise to any cell lineage we want. SCNT (Stem Cell Nuclear Transport) is used for developing embryonic stem cells (ESC) that will have both research and therapeutic application. ESC are created by removing nucleus from the egg and implanting nucleus from adult somatic cell (containing both mother and father genetic material). Egg will act like it’s been fertilized and start dividing first to reach blastocyst stage and then toward any cell lineage we want. Procedure is the same with animal species and could be used to produce additional food source (by cloning farm animals) or to prevent extinction of endangered animals. It may sound like simple process, but success rate with this kind of genetic manipulation is pretty low. Dolly the sheep was first mammal created in laboratory. Out of 277 eggs used for SCNT, just 29 embryos were created. 3 survived until birth and only one - more famous by its given name – Dolly, survived until adulthood. Although, genetic material in the newly formed cell (organism) is the same as in donor’s cell, certain part of DNA is unique. Each cell contains mitochondria with its own genetic material. It’s inherited solely from the mother due to couple of reasons: egg contains more mtDNA than sperm; sperm derived mtDNA is easily degraded once inside or it can even fail to enter the egg. Thanks to this phenomenon, cloned cells can be considered genetic hybrids, as they contain both somatic DNA and mother mitochondrial genes.

Some large animals can create clones on their own. Lizards, snakes, ants, crustaceous species and even certain sharks are able to produce new individual by parthenogenesis – out of unfertilized egg. For most species, this is not obligatory way to reproduce but a method to overcome crisis in their environment. Komodo dragon, for example, can reproduce by parthenogenesis to increase the population in the habitat and then switch back to sexual reproduction to increase genetic diversity of the next generation.

To conclude, cloning is not something we invented, it’s natural phenomenon that we start exploiting recently. Wise and careful approach could be beneficial for the planet; we just need to pay attention not to cross the line, as genetic diversity is what allowed us to survive so far.]]> https://www.biotechnologyforums.com/thread-1775.html Fri, 12 Oct 2012 11:20:50 +0000 priyasaravanan_1406]]> https://www.biotechnologyforums.com/thread-1775.html
Transformation: Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. This single stranded DNA mingles with the host genetic material successfully.

The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation. In the chemical mediated gene transfer, the cold conditioned cells in calcium chloride solution are exposed to sudden heat which increases the permeability of the cell membrane allowing the foreign DNA. The electroporation method as the name indicates, pores are made in the cell by exposing it to suitable electric field, allowing the entry of the DNA. The opened up portions of the cell are sealed by the ability of the cell to repair.

Transduction: In transduction, a media like virus is required between two bacterial cells in transferring genes from one cell to the other. Researchers used virus as a tool to introduce foreign DNA from the selected species to the target organism. Transduction mode of gene transfer follows either a lysogenic phase or lytic phase. In the lysogenic phase, the viral (phage) DNA once joining the bacterial DNA through transduction stays dormant in the following generations. The induction of lysogenic cycle by an external factor like UV light results in lytic phase. In lytic phase, the viral or phage DNA exists a s a separate entity in the host cell and the host cell replicates viral DNA mistaking it for its own DNA.As a result many phages are produced within the host cell and when the number exceeds it causes the lysis of the host cell and the phages exits and infects other cells. As this process involves existence of both the genome of the phage and the genome of the bacteria in the same cell, it may result in exchange of some genes between the two DNA. As a result, the newly developed phage leaving the cell may carry a bacterial gene and transfer it to the other cell it infects. Also some of the phage genes may be present in the host cell. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. The significance of creating pores and introducing the DNA into the host mammalian cell contributed to different methods in transfection. Chemical mediated transfection involves use of either calcium phosphate or cationic polymers or liposomes. Electroporation, sonoporation, impalefection, optical transfection, hydro dynamic delivery are some of the non chemical based gene transfer. Particle based transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to host cell or by another method called as magnetofection. Nucleofection and use of heat shock are the other evolved methods for successful transfection.]]>
Transformation: Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. This single stranded DNA mingles with the host genetic material successfully.

The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation. In the chemical mediated gene transfer, the cold conditioned cells in calcium chloride solution are exposed to sudden heat which increases the permeability of the cell membrane allowing the foreign DNA. The electroporation method as the name indicates, pores are made in the cell by exposing it to suitable electric field, allowing the entry of the DNA. The opened up portions of the cell are sealed by the ability of the cell to repair.

Transduction: In transduction, a media like virus is required between two bacterial cells in transferring genes from one cell to the other. Researchers used virus as a tool to introduce foreign DNA from the selected species to the target organism. Transduction mode of gene transfer follows either a lysogenic phase or lytic phase. In the lysogenic phase, the viral (phage) DNA once joining the bacterial DNA through transduction stays dormant in the following generations. The induction of lysogenic cycle by an external factor like UV light results in lytic phase. In lytic phase, the viral or phage DNA exists a s a separate entity in the host cell and the host cell replicates viral DNA mistaking it for its own DNA.As a result many phages are produced within the host cell and when the number exceeds it causes the lysis of the host cell and the phages exits and infects other cells. As this process involves existence of both the genome of the phage and the genome of the bacteria in the same cell, it may result in exchange of some genes between the two DNA. As a result, the newly developed phage leaving the cell may carry a bacterial gene and transfer it to the other cell it infects. Also some of the phage genes may be present in the host cell. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. The significance of creating pores and introducing the DNA into the host mammalian cell contributed to different methods in transfection. Chemical mediated transfection involves use of either calcium phosphate or cationic polymers or liposomes. Electroporation, sonoporation, impalefection, optical transfection, hydro dynamic delivery are some of the non chemical based gene transfer. Particle based transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to host cell or by another method called as magnetofection. Nucleofection and use of heat shock are the other evolved methods for successful transfection.]]> https://www.biotechnologyforums.com/thread-1747.html Mon, 08 Oct 2012 13:33:27 +0000 priyasaravanan_1406]]> https://www.biotechnologyforums.com/thread-1747.html
Monoclonal antibody is the term used for the antibody produced in vitro by multiplying a single hybrid cell, obtained by cloning selected cells from a single source. Monoclonal antibodies are known for its purity and specificity. The conventional method of monoclonal antibody production was done by injecting the test animal with a particular type of antigen. After few days of the dose of the antigen, blood is drawn from the test animal and the antibodies were extracted from the serum of the blood. This method was failure both qualitatively and quantitatively. The antibodies obtained were found to be impure (mixed variants) and the amount obtained was also significantly less. Hence adoption of cloning technique was identified as the optional method to produce antibodies in vitro.

In this method, scientists selected tumor cells for its ability to multiply intensively and the antibody producing mammalian cells and fused these two under in vitro conditions. On the onset of the production, the test animal usually a mice is injected with an antigen to stimulate the antibody production. The antibody producing cells are identified and extracted from the spleen of the mice and it is fused with the myeloma cells which were extracted from the mice earlier and cultured in vitro. The resulting hybrid cell or hybridoma is observed for the presence of the desired antibody and once satisfied, the hybrids are subjected to grow in culture to produce splendid quantity of monoclonal antibodies. Again, the extraction and purification of the monoclonal antibody from the hybridoma is done by sequence of processes like centrifugation, filtration, ultra filtration or dialysis and ion exchange chromatography. Later the ion exchange chromatography was replaced by size exclusion chromatography which was found to be more effective in purifying. Also a procedure called affinity purification was employed to obtain the maximum purity. After undergoing the steps of purification, the final product, the monoclonal antibody is checked for the level of purity by using either chromatogram or gel electrophoresis or capillary electrophoresis.

The first test animal used for the production of monoclonal antibody is mice and a consequence reaction like allergy was observed in humans when supplemented with the monoclonal antibodies produced from mouse cell. Also, humans responded only to the initial dose and developed resistance to further doses. This posed as a bigger problem in obtaining the benefits of the monoclonal antibody and as a result evolved the chimeric antibody. The chimeric antibody is developed by inserting some human amino acid sequence into the animal developed monoclonal antibody.

The novel idea of developing fully human monoclonal antibody is a major breakthrough in the production of monoclonal antibody. In this method, blood sample is collected from an individual (donor) recovered from a particular type of infection and the antibody specific cells are extracted and immortalized. These cells are then subjected to micro well assay technique and the antigen specific antibodies are identified by fluorescence method and isolated. These cells are expanded and characterized before passing to other cell types for large scale production. The difficulty in identifying a donor is eluded by extracting cell from a healthy person and activating the cell for specific antibody production in vitro. The advancement in genetic engineering technology serves the human monoclonal antibody production by using transgenic mice.

The wide therapeutic application of monoclonal antibodies states the significance of the production of the monoclonal antibody.]]>
Monoclonal antibody is the term used for the antibody produced in vitro by multiplying a single hybrid cell, obtained by cloning selected cells from a single source. Monoclonal antibodies are known for its purity and specificity. The conventional method of monoclonal antibody production was done by injecting the test animal with a particular type of antigen. After few days of the dose of the antigen, blood is drawn from the test animal and the antibodies were extracted from the serum of the blood. This method was failure both qualitatively and quantitatively. The antibodies obtained were found to be impure (mixed variants) and the amount obtained was also significantly less. Hence adoption of cloning technique was identified as the optional method to produce antibodies in vitro.

In this method, scientists selected tumor cells for its ability to multiply intensively and the antibody producing mammalian cells and fused these two under in vitro conditions. On the onset of the production, the test animal usually a mice is injected with an antigen to stimulate the antibody production. The antibody producing cells are identified and extracted from the spleen of the mice and it is fused with the myeloma cells which were extracted from the mice earlier and cultured in vitro. The resulting hybrid cell or hybridoma is observed for the presence of the desired antibody and once satisfied, the hybrids are subjected to grow in culture to produce splendid quantity of monoclonal antibodies. Again, the extraction and purification of the monoclonal antibody from the hybridoma is done by sequence of processes like centrifugation, filtration, ultra filtration or dialysis and ion exchange chromatography. Later the ion exchange chromatography was replaced by size exclusion chromatography which was found to be more effective in purifying. Also a procedure called affinity purification was employed to obtain the maximum purity. After undergoing the steps of purification, the final product, the monoclonal antibody is checked for the level of purity by using either chromatogram or gel electrophoresis or capillary electrophoresis.

The first test animal used for the production of monoclonal antibody is mice and a consequence reaction like allergy was observed in humans when supplemented with the monoclonal antibodies produced from mouse cell. Also, humans responded only to the initial dose and developed resistance to further doses. This posed as a bigger problem in obtaining the benefits of the monoclonal antibody and as a result evolved the chimeric antibody. The chimeric antibody is developed by inserting some human amino acid sequence into the animal developed monoclonal antibody.

The novel idea of developing fully human monoclonal antibody is a major breakthrough in the production of monoclonal antibody. In this method, blood sample is collected from an individual (donor) recovered from a particular type of infection and the antibody specific cells are extracted and immortalized. These cells are then subjected to micro well assay technique and the antigen specific antibodies are identified by fluorescence method and isolated. These cells are expanded and characterized before passing to other cell types for large scale production. The difficulty in identifying a donor is eluded by extracting cell from a healthy person and activating the cell for specific antibody production in vitro. The advancement in genetic engineering technology serves the human monoclonal antibody production by using transgenic mice.

The wide therapeutic application of monoclonal antibodies states the significance of the production of the monoclonal antibody.]]> https://www.biotechnologyforums.com/thread-60.html Sun, 19 Sep 2010 06:14:13 +0000 NatashaKundi]]> https://www.biotechnologyforums.com/thread-60.html
Method of Therapeutic Cloning:-
Method of therapeutic cloning is used to treat the patient’s tissues and organs which have got damaged due to some disease or any other disorder. In this method patient’s own skin cells are used so that the immune system can easily accept the newly developed tissues or organs. There is a particular method through which therapeutic cloning is done.

1) First the DNA of the woman’s ovum is removed which is the basic of human life.

2) Take the DNA of the desired cell like skin cell and insert it into the ovum of the woman.

3) To insert the foreign DNA into the ovum, an electric shock is given so that embryo starts to develop. Due to the shock, a pre-embryo will produce.

4) The cells of the pre-embryo keep developing until they start producing stem cells.

5) When the stem cells are produced, they are taken out of the embryo which results in the death of the embryo.

6) Stem cells are allowed to grow in a specific culture where they will produce desired tissues and organs. Stem cells have the ability to develop into various forms of tissues and organs.

7) The developed tissues or organs can be transferred into the patient who suffers from the diseases like diabetes or Alzheimer’s.

Benefits of Therapeutic Cloning:-
Therapeutic cloning is helpful in creating the replacement organs. Patients who are suffering from kidney disorders or other disorders like this. It is beneficial in giving people more years to enjoy their life. When the patients own body cells are used in therapeutic cloning, then the fear of organ or tissue rejection by immune system vanishes and doctors can easily replace the organs. In some countries where this technique has not yet been applied, patients wait for the required organ for many days and in this period their life also come in danger, if therapeutic cloning is applied, the watt for the right organ will come to a stop and patients will easily get the required organ.

As there is a custom that a person donates his own organ for the needy patient, but now patient will be able to get a brand new organ just because of therapeutic cloning? It may be helpful in curing the disease which is difficult to cure by other means.

Disadvantages of therapeutic cloning:-
Though therapeutic cloning is a promising technique for the organ transplantation but it is not applicable in adult stem cells. As the embryo develops into a complete human being so when the stem cells are taken from the embryo and it dies as a result, people consider it a murder. Therapeutic cloning may develop tumors or other diseases in the body. The most important concern of therapeutic cloning is where to get the egg from. In my view no woman will be willing to donate her egg.]]>
Method of Therapeutic Cloning:-
Method of therapeutic cloning is used to treat the patient’s tissues and organs which have got damaged due to some disease or any other disorder. In this method patient’s own skin cells are used so that the immune system can easily accept the newly developed tissues or organs. There is a particular method through which therapeutic cloning is done.

1) First the DNA of the woman’s ovum is removed which is the basic of human life.

2) Take the DNA of the desired cell like skin cell and insert it into the ovum of the woman.

3) To insert the foreign DNA into the ovum, an electric shock is given so that embryo starts to develop. Due to the shock, a pre-embryo will produce.

4) The cells of the pre-embryo keep developing until they start producing stem cells.

5) When the stem cells are produced, they are taken out of the embryo which results in the death of the embryo.

6) Stem cells are allowed to grow in a specific culture where they will produce desired tissues and organs. Stem cells have the ability to develop into various forms of tissues and organs.

7) The developed tissues or organs can be transferred into the patient who suffers from the diseases like diabetes or Alzheimer’s.

Benefits of Therapeutic Cloning:-
Therapeutic cloning is helpful in creating the replacement organs. Patients who are suffering from kidney disorders or other disorders like this. It is beneficial in giving people more years to enjoy their life. When the patients own body cells are used in therapeutic cloning, then the fear of organ or tissue rejection by immune system vanishes and doctors can easily replace the organs. In some countries where this technique has not yet been applied, patients wait for the required organ for many days and in this period their life also come in danger, if therapeutic cloning is applied, the watt for the right organ will come to a stop and patients will easily get the required organ.

As there is a custom that a person donates his own organ for the needy patient, but now patient will be able to get a brand new organ just because of therapeutic cloning? It may be helpful in curing the disease which is difficult to cure by other means.

Disadvantages of therapeutic cloning:-
Though therapeutic cloning is a promising technique for the organ transplantation but it is not applicable in adult stem cells. As the embryo develops into a complete human being so when the stem cells are taken from the embryo and it dies as a result, people consider it a murder. Therapeutic cloning may develop tumors or other diseases in the body. The most important concern of therapeutic cloning is where to get the egg from. In my view no woman will be willing to donate her egg.]]> https://www.biotechnologyforums.com/thread-59.html Sun, 19 Sep 2010 06:10:50 +0000 NatashaKundi]]> https://www.biotechnologyforums.com/thread-59.html
Steps Involved in PCR:-
To initiate the polymerase chain reaction it is necessary to have some things like DNA polymerases, Restriction enzymes, a DNA template, primers or short DNA sequences to start the DNA polymerase. Without which the task of making multiple copies cannot be accomplished.

Denaturation:-
The reaction mixture is heated at the temperature of 94 to 98 degree Celsius for 20 to 30 minutes. It makes the double stranded DNA to melt by breaking the hydrogen bonds that keep the two templates together. Now DNA molecule becomes single stranded.

Annealing:-
After the separation of the DNA strands, temperature of the reaction mixture is lowered and is kept at 50 to 65 degree Celsius for 20 to 40 seconds. It causes the annealing of the primers to the single stranded DNA molecules.

Extension:-
In this step, the DNA polymerase makes a complementary strand against each single stranded DNA. Usually Taq polymerase is used for this purpose.

Elongation:-
Usually a temperature of 70 to 74 degree Celsius is required for this step to make sure that all the single stranded DNA templates have found their complementary strands.

Uses of PCR:-
The use of polymerase chain reaction is important in many scientific fields like genetics, genetic engineering and molecular biology. In the disciplines like microbiology and molecular biology, it is used for DNA cloning procedures, DNA sequencing, and recombinant DNA technology in the research laboratories. In clinical microbiology, many microbial infections are diagnosed by PCR. Similarly, epidemiological studies also make use of this technique. Field of forensics is another important field which uses PCR for the identification of criminals and to identify the paternity of the child in the court of justice by obtaining only small amount of blood.

Importance of PCR in Biotechnology:-
Polymerase chain reaction is of vital importance in biotechnology. It can be used for the diagnosis of diseases like AIDS, middle ear infection, Lyme disease and tuberculosis. PCR identifies and cultures those microorganisms which are responsible for causing these diseases. This technique also helped a lot during human genome project in the isolation, amplification and sequencing of human genes. Recombinant DNA technology also makes use of the PCR for making multiple copies of transgenes for different applications for example for diagnosing the diseases and for making certain changes in the genome of an organism.

Disadvantages of PCR:-
Like any other reactions, reaction occurring in the PCR also faces some problems. Usually the polymerases used in PCR do not contain the 3’ to 5’ exonuclease activity. Due to this reason, they are not able to correct any errors while incorporating the nucleotides.]]>
Steps Involved in PCR:-
To initiate the polymerase chain reaction it is necessary to have some things like DNA polymerases, Restriction enzymes, a DNA template, primers or short DNA sequences to start the DNA polymerase. Without which the task of making multiple copies cannot be accomplished.

Denaturation:-
The reaction mixture is heated at the temperature of 94 to 98 degree Celsius for 20 to 30 minutes. It makes the double stranded DNA to melt by breaking the hydrogen bonds that keep the two templates together. Now DNA molecule becomes single stranded.

Annealing:-
After the separation of the DNA strands, temperature of the reaction mixture is lowered and is kept at 50 to 65 degree Celsius for 20 to 40 seconds. It causes the annealing of the primers to the single stranded DNA molecules.

Extension:-
In this step, the DNA polymerase makes a complementary strand against each single stranded DNA. Usually Taq polymerase is used for this purpose.

Elongation:-
Usually a temperature of 70 to 74 degree Celsius is required for this step to make sure that all the single stranded DNA templates have found their complementary strands.

Uses of PCR:-
The use of polymerase chain reaction is important in many scientific fields like genetics, genetic engineering and molecular biology. In the disciplines like microbiology and molecular biology, it is used for DNA cloning procedures, DNA sequencing, and recombinant DNA technology in the research laboratories. In clinical microbiology, many microbial infections are diagnosed by PCR. Similarly, epidemiological studies also make use of this technique. Field of forensics is another important field which uses PCR for the identification of criminals and to identify the paternity of the child in the court of justice by obtaining only small amount of blood.

Importance of PCR in Biotechnology:-
Polymerase chain reaction is of vital importance in biotechnology. It can be used for the diagnosis of diseases like AIDS, middle ear infection, Lyme disease and tuberculosis. PCR identifies and cultures those microorganisms which are responsible for causing these diseases. This technique also helped a lot during human genome project in the isolation, amplification and sequencing of human genes. Recombinant DNA technology also makes use of the PCR for making multiple copies of transgenes for different applications for example for diagnosing the diseases and for making certain changes in the genome of an organism.

Disadvantages of PCR:-
Like any other reactions, reaction occurring in the PCR also faces some problems. Usually the polymerases used in PCR do not contain the 3’ to 5’ exonuclease activity. Due to this reason, they are not able to correct any errors while incorporating the nucleotides.]]> https://www.biotechnologyforums.com/thread-37.html Fri, 16 Jul 2010 00:01:48 +0000 NatashaKundi]]> https://www.biotechnologyforums.com/thread-37.html What is Cloning?
It is a process by which an identical copy of DNA is produced. It is used to reproduce type specific cells such as an individual organism like Dolly Sheep. Unlike normal reproduction, cloning requires a single parent. A few types of plants have been cloned in the course of many years but cloning of animals especially humans is an ethical issue. Cloning enables illnesses to be cured and to make plants healthier.

Cloning is done by using a circular DNA as a carrier in which a foreign piece of DNA is put. Special genetic information is held within this foreign piece of DNA. The circular DNA is called a vector which acts as a means of transport. The vector then replicates and is then transferred from one living organism to another. This living organism is called a host as its body acts as a temporary home for this DNA and its vector. The new circular DNA with the foreign piece is called a clone which is different from the parental vector.

Nuclear Transfer is a type of cloning that involves transfer of nucleus from a somatic cell (e.g. blood cell, heart cell) to an egg cell. The nucleus of the somatic cell is removed by a micropipette and injected into an enucleated oocyte, which is an unfertilized cell. The nucleus is then given an electric shock so as to fuse with the cytoplasm. It then divides until it becomes an embryo and is then implanted into a surrogate thus giving rise to an identical organism.

Artificial Twinning is also a type of cloning in which an embryo is artificially divided into two or more embryos. An egg is fertilized by sperm. In the early stages of its formation, the embryo is split into two or more embryos which are then left to grow in a surrogate. The offspring thus produced are identical.

Before the birth of Dolly Sheep, an experiment was performed on frog cells. Nucleus from a frog’s gut cell was injected into an enucleated oocyte. The cell began to divide until an embryo was formed thus resulting in tadpoles. Earlier on, this technique only worked on frogs. Scientists could not clone mammals. Another drawback was that the tadpoles never grew into frogs; they died at a very early stage.

After a lot of experimentation, scientists discovered that using a quiescent (undividable) cell instead of a fast dividing cell will prove helpful in cloning of mammals. Therefore they started an experiment on sheep. The method used was Nuclear Transfer. They took a cell from the mammary glands of a Finn Dorset breed of sheep, removed the nucleus and injected it into a Blackface ewe breed. An electric pulse was then used to fuse the nucleus with the cytoplasm of the cell. This fused cell was then transferred into a Blackface ewe. This process was repeated 276 times and after 148 days, Dolly Sheep was born on 5th July 1996. This success enabled scientists to clone domestic animals such as cows, horses, bulls etc. After being bred with a Welsh Mountain ram, Dolly gave birth to six lambs. She then died at the age of six as she developed lung diseases and arthritis.]]> What is Cloning?
It is a process by which an identical copy of DNA is produced. It is used to reproduce type specific cells such as an individual organism like Dolly Sheep. Unlike normal reproduction, cloning requires a single parent. A few types of plants have been cloned in the course of many years but cloning of animals especially humans is an ethical issue. Cloning enables illnesses to be cured and to make plants healthier.

Cloning is done by using a circular DNA as a carrier in which a foreign piece of DNA is put. Special genetic information is held within this foreign piece of DNA. The circular DNA is called a vector which acts as a means of transport. The vector then replicates and is then transferred from one living organism to another. This living organism is called a host as its body acts as a temporary home for this DNA and its vector. The new circular DNA with the foreign piece is called a clone which is different from the parental vector.

Nuclear Transfer is a type of cloning that involves transfer of nucleus from a somatic cell (e.g. blood cell, heart cell) to an egg cell. The nucleus of the somatic cell is removed by a micropipette and injected into an enucleated oocyte, which is an unfertilized cell. The nucleus is then given an electric shock so as to fuse with the cytoplasm. It then divides until it becomes an embryo and is then implanted into a surrogate thus giving rise to an identical organism.

Artificial Twinning is also a type of cloning in which an embryo is artificially divided into two or more embryos. An egg is fertilized by sperm. In the early stages of its formation, the embryo is split into two or more embryos which are then left to grow in a surrogate. The offspring thus produced are identical.

Before the birth of Dolly Sheep, an experiment was performed on frog cells. Nucleus from a frog’s gut cell was injected into an enucleated oocyte. The cell began to divide until an embryo was formed thus resulting in tadpoles. Earlier on, this technique only worked on frogs. Scientists could not clone mammals. Another drawback was that the tadpoles never grew into frogs; they died at a very early stage.

After a lot of experimentation, scientists discovered that using a quiescent (undividable) cell instead of a fast dividing cell will prove helpful in cloning of mammals. Therefore they started an experiment on sheep. The method used was Nuclear Transfer. They took a cell from the mammary glands of a Finn Dorset breed of sheep, removed the nucleus and injected it into a Blackface ewe breed. An electric pulse was then used to fuse the nucleus with the cytoplasm of the cell. This fused cell was then transferred into a Blackface ewe. This process was repeated 276 times and after 148 days, Dolly Sheep was born on 5th July 1996. This success enabled scientists to clone domestic animals such as cows, horses, bulls etc. After being bred with a Welsh Mountain ram, Dolly gave birth to six lambs. She then died at the age of six as she developed lung diseases and arthritis.]]>