11-16-2012, 10:31 PM
(This post was last modified: 11-16-2012, 11:21 PM by Administrator.)
The whole genetic constitution of an organism is known as genome of an organism. A detailed description of positional, structural and functional properties of the entire genome is known as genome maps. It can be broadly classified as cytogenic and molecular genome maps depending on the position of the genes and the total information of the gene respectively.
An initiative was started by many countries to obtain entire information about the genome of humans. This is known as human genome project. This was started in the year of 1990 and a complete draft was submitted in 2003. The strategy involved in human genome project was:
(i) Preparing maps of each gene of the genome to decide the location of the gene
(ii) Sequencing the genes to determine the different gene elements and any variations in the same within or between two genomes
(iii) Functional analysis of genes to determine the role of each gene in an organism.
The major markers involved in the genome projects are RAPD, RFLP, VNTR, chromosome jumping etc. This helps in mapping and sequencing of unknown genes.
Restriction fragment length Polymorphism (RFLP): This employs a mechanism by which same genes of different organisms or related organisms can be studied. The mode of action involves restriction digestion with a specific restriction enzyme. Genomic DNA is isolated from different species or related organisms. This DNA is digested with the help of a selected restriction enzyme and the fragments produced are separated through gel electrophoresis. This gel is transferred onto a solid support and is followed by hybridization of the gel with appropriate radio-labelled DNA probes. The gel is then scanned by auto radiography. The pattern generated by different organisms differs in these patterns and mainly depends upon the DNA used for digestion, the restriction enzyme used and the DNA probe used. The difference in the genomic DNA accounting to various RFLP is the changes in the base recognition sequences of the restriction enzyme, or addition or deletions. The probes used is available from a variety of genomic library, chromosome specific library etc, based on the bands available on the gels, each organism has a specific RFLP loci for specific restriction enzyme and an RFLP map is created. This technique helps in mapping even very small segments of DNA and the process is very rapid but the disadvantages like high cost and the need of skilled personnel.
Random Amplified Polymorphic DNA (RAPD): This technique mainly employs PCR. In this, a genomic DNA is isolated and fed into a PCR. Here, the process of denaturation leads to unwinding of the two strands of DNA. The denatured strands when renatured is added with a short oligonucleotide sequence called primer which is of known sequence. Upon annealing this sequence pairs with the homologous sequences in the DNA which has similar sequences at random locations. Sequences in DNA complimentary to primer at the both ends are amplified during the amplification process. The amplified DNA is detected by gel electrophoresis followed by fluorescence detection.
Since this process does not utilize restriction enzymes and probes it is comparatively cost effective. But the reproducibility in comparison to RFLP is poor.
Chromosome walking:
This is a chromosome based technique and helps in obtaining detailed knowledge of chromosome. It requires knowledge of a genetic marker. This known gene marker is used to identify a clone that has a corresponding DNA insert. The DNA fragment consisting of such a known marker is isolated and a restriction map of this fragment is prepared. This is followed by isolation and sub cloning of a small segment of this fragment. This clone acts as a probe in identifying the presence of such segment in a genome library. The gene identified in this manner will have one end of the segment similar to the probe and an unknown fragment. A restriction map of this unknown fragment is prepared and similarly used to probe identical sequences in another library. The resulting probing will involve production of new fragment with one known and one unknown sequence. This process can be continued until the end of chromosome is reached. Thus it helps in ‘walking’ of the chromosome.
Variable number of tandem repeats (VNTR): This consists of regions in DNA which has variable number of repeats. Different individuals show different repeats at a given loci or position of a chromosome. This constitutes alleles of VNTR loci. They can be broadly classified as micro and mini satellite DNAs. These make up the hyper variable region of DNA. Minisatellite DNA consists of pro terminal regions of DNA. Micro satellite DNA consist of regions which are short sequences and more frequent hence represent an efficient marker system.
Thus it is possible, with the help of such markers to sequence and map efficiently genetic constitution of an organism as a whole resulting in developing genome map of the organism.
An initiative was started by many countries to obtain entire information about the genome of humans. This is known as human genome project. This was started in the year of 1990 and a complete draft was submitted in 2003. The strategy involved in human genome project was:
(i) Preparing maps of each gene of the genome to decide the location of the gene
(ii) Sequencing the genes to determine the different gene elements and any variations in the same within or between two genomes
(iii) Functional analysis of genes to determine the role of each gene in an organism.
The major markers involved in the genome projects are RAPD, RFLP, VNTR, chromosome jumping etc. This helps in mapping and sequencing of unknown genes.
Restriction fragment length Polymorphism (RFLP): This employs a mechanism by which same genes of different organisms or related organisms can be studied. The mode of action involves restriction digestion with a specific restriction enzyme. Genomic DNA is isolated from different species or related organisms. This DNA is digested with the help of a selected restriction enzyme and the fragments produced are separated through gel electrophoresis. This gel is transferred onto a solid support and is followed by hybridization of the gel with appropriate radio-labelled DNA probes. The gel is then scanned by auto radiography. The pattern generated by different organisms differs in these patterns and mainly depends upon the DNA used for digestion, the restriction enzyme used and the DNA probe used. The difference in the genomic DNA accounting to various RFLP is the changes in the base recognition sequences of the restriction enzyme, or addition or deletions. The probes used is available from a variety of genomic library, chromosome specific library etc, based on the bands available on the gels, each organism has a specific RFLP loci for specific restriction enzyme and an RFLP map is created. This technique helps in mapping even very small segments of DNA and the process is very rapid but the disadvantages like high cost and the need of skilled personnel.
Random Amplified Polymorphic DNA (RAPD): This technique mainly employs PCR. In this, a genomic DNA is isolated and fed into a PCR. Here, the process of denaturation leads to unwinding of the two strands of DNA. The denatured strands when renatured is added with a short oligonucleotide sequence called primer which is of known sequence. Upon annealing this sequence pairs with the homologous sequences in the DNA which has similar sequences at random locations. Sequences in DNA complimentary to primer at the both ends are amplified during the amplification process. The amplified DNA is detected by gel electrophoresis followed by fluorescence detection.
Since this process does not utilize restriction enzymes and probes it is comparatively cost effective. But the reproducibility in comparison to RFLP is poor.
Chromosome walking:
This is a chromosome based technique and helps in obtaining detailed knowledge of chromosome. It requires knowledge of a genetic marker. This known gene marker is used to identify a clone that has a corresponding DNA insert. The DNA fragment consisting of such a known marker is isolated and a restriction map of this fragment is prepared. This is followed by isolation and sub cloning of a small segment of this fragment. This clone acts as a probe in identifying the presence of such segment in a genome library. The gene identified in this manner will have one end of the segment similar to the probe and an unknown fragment. A restriction map of this unknown fragment is prepared and similarly used to probe identical sequences in another library. The resulting probing will involve production of new fragment with one known and one unknown sequence. This process can be continued until the end of chromosome is reached. Thus it helps in ‘walking’ of the chromosome.
Variable number of tandem repeats (VNTR): This consists of regions in DNA which has variable number of repeats. Different individuals show different repeats at a given loci or position of a chromosome. This constitutes alleles of VNTR loci. They can be broadly classified as micro and mini satellite DNAs. These make up the hyper variable region of DNA. Minisatellite DNA consists of pro terminal regions of DNA. Micro satellite DNA consist of regions which are short sequences and more frequent hence represent an efficient marker system.
Thus it is possible, with the help of such markers to sequence and map efficiently genetic constitution of an organism as a whole resulting in developing genome map of the organism.
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