10-25-2012, 06:05 PM
(This post was last modified: 10-25-2012, 07:07 PM by Administrator.)
DNA Microarrays consist of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes.
After a gene has been isolated, the next step involved is to study the different characteristics of the isolated gene. In the past, it was possible to analyse the nature and properties of only a single gene but with the advent of microarray technology it is possible to analyse thousands of gene with a single experiment resulting in a faster and more accurate results.
Microarray: It refers to a recent hybridization technique which provides an opportunity to match known and unknown DNA samples under specific conditions to reveal different properties of the unknown gene. Here, a series of probes is fixed onto a solid substrate and used to hybridize a series of test DNA. It is of two types DNA microarray and antibody microarrays.
DNA microarrays (DNA chips, bio chip, gene chip):
This consists of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes etc. In a more recent technique, an array of microscopic beads is used as a platform for such arrays.
Probe and Target:
A probe refers to a small nucleotide sequences with known bases. This is fixed onto a solid substrate to form a neat arrangement called an array. Purified mRNA, isolated DNA, cDNA produced from mRNA; all can form probes.
Target refers to test a DNA sequence which has to be studied for its position, composition, gene expression, mutations etc; these are labelled using fluorochrome or other labels which aid in quantifying the results after hybridization.
DNA microarrays are of two different types
cDNA arrays:
The probes consisting of complementary DNA is spotted onto the glass substrate with the help of fine needles and a robotic arm.
Oligonucleotide arrays:
The probes here are oligonucleotides formulated in situ or produced externally and then immobilized on the arrays by different techniques like photolithography.
Earlier the oligonucleotide array was termed as DNA chips. But recently the term DNA or gene chips are applicable to both the kind of arrays.
Principle and mechanism:
In order to conduct such a study, the complementary base pairing nature of the polynucleotide chain of the DNA and the resulting double helix plays an important role. In the process of hybridization, these polynucleotides are subjected to conditions so that the hydrogen bonds between the two stands weaken but phoshodiester bonds between the nucleotides remains intact leading to production of two separate polynucleotide strands. When treated with appropriate probes, it results in the pairing of the probe with its complementary bases in the target DNA.
The DNA sequences of each gene to be analysed of an organism are labelled with fluorochrome like Cys green or red. DNA micro array is set up by spotting or other available techniques. When such a probe is treated with the labelled target DNA and hybridization is initiated, the double helix open up and hybridize with complementary DNA resulting in producing fluorescence at hybridized sites. These are scanned and quantified. Different fluorochrome representing different properties can be used to be probed with a single DNA chip resulting in conducting multiple tests using a single microarray.
Multiple and single channel microarrays:
In a multiple channel or two colour microarray, it is possible to analyse genes from different sample in a single test. Each target gene is labelled with fluorochrome having different fluorescence emission. This mixed sample is allowed to hybridize in a single test with a single microarray probe, and when scanned with microarray scanner after excitement with different corresponding wavelengths, the ratio of different gene can be quantified.
In a single channel or one colour array, a probe is hybridized with target DNA labelled with one colour fluorochrome. Two colour microarrays is usually used to detect different genes present in one mixed sample whereas one colour microarray usually is employed to estimate the amount of gene expression of same sample or between samples. Single channel microarrays are found to give more accurate results but need to conduct different test to quantify different gene expression. Multi channel makes use of only one test to give multiple results but different samples sometimes interfere with each other leading to non unique results.
Applications:
(i)Diagnostic applications:
Micro array technology can be used to detect presence of genetic diseases, presence of mutations, polymorphism, cancer etc.
(ii)Gene expression profiling:
Using this technique, the number of genes present, the expression rate each gene can be determined. Monitoring the gene expression is known as gene expression profiling. Quantitative and qualitative measurements of such expressions is possible with the help of microarrays.
(iii)Genomics:
With the help of microarrays, SNPs or single nucleotide polymorphisms can be detected. It refers to different nucleotides present at the same base position in different alleles. Such minute differences between different individuals or different alleles of same gene can be identified successfully and uniquely with the help of micro arrays.
(iv)Sequencing:
A microarray is prepared with oligonucleotides of specific length. When hybridized with target sequences of unknown lengths, the results obtained indicate the length of the target sequence.
After a gene has been isolated, the next step involved is to study the different characteristics of the isolated gene. In the past, it was possible to analyse the nature and properties of only a single gene but with the advent of microarray technology it is possible to analyse thousands of gene with a single experiment resulting in a faster and more accurate results.
Microarray: It refers to a recent hybridization technique which provides an opportunity to match known and unknown DNA samples under specific conditions to reveal different properties of the unknown gene. Here, a series of probes is fixed onto a solid substrate and used to hybridize a series of test DNA. It is of two types DNA microarray and antibody microarrays.
DNA microarrays (DNA chips, bio chip, gene chip):
This consists of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes etc. In a more recent technique, an array of microscopic beads is used as a platform for such arrays.
Probe and Target:
A probe refers to a small nucleotide sequences with known bases. This is fixed onto a solid substrate to form a neat arrangement called an array. Purified mRNA, isolated DNA, cDNA produced from mRNA; all can form probes.
Target refers to test a DNA sequence which has to be studied for its position, composition, gene expression, mutations etc; these are labelled using fluorochrome or other labels which aid in quantifying the results after hybridization.
DNA microarrays are of two different types
cDNA arrays:
The probes consisting of complementary DNA is spotted onto the glass substrate with the help of fine needles and a robotic arm.
Oligonucleotide arrays:
The probes here are oligonucleotides formulated in situ or produced externally and then immobilized on the arrays by different techniques like photolithography.
Earlier the oligonucleotide array was termed as DNA chips. But recently the term DNA or gene chips are applicable to both the kind of arrays.
Principle and mechanism:
In order to conduct such a study, the complementary base pairing nature of the polynucleotide chain of the DNA and the resulting double helix plays an important role. In the process of hybridization, these polynucleotides are subjected to conditions so that the hydrogen bonds between the two stands weaken but phoshodiester bonds between the nucleotides remains intact leading to production of two separate polynucleotide strands. When treated with appropriate probes, it results in the pairing of the probe with its complementary bases in the target DNA.
The DNA sequences of each gene to be analysed of an organism are labelled with fluorochrome like Cys green or red. DNA micro array is set up by spotting or other available techniques. When such a probe is treated with the labelled target DNA and hybridization is initiated, the double helix open up and hybridize with complementary DNA resulting in producing fluorescence at hybridized sites. These are scanned and quantified. Different fluorochrome representing different properties can be used to be probed with a single DNA chip resulting in conducting multiple tests using a single microarray.
Multiple and single channel microarrays:
In a multiple channel or two colour microarray, it is possible to analyse genes from different sample in a single test. Each target gene is labelled with fluorochrome having different fluorescence emission. This mixed sample is allowed to hybridize in a single test with a single microarray probe, and when scanned with microarray scanner after excitement with different corresponding wavelengths, the ratio of different gene can be quantified.
In a single channel or one colour array, a probe is hybridized with target DNA labelled with one colour fluorochrome. Two colour microarrays is usually used to detect different genes present in one mixed sample whereas one colour microarray usually is employed to estimate the amount of gene expression of same sample or between samples. Single channel microarrays are found to give more accurate results but need to conduct different test to quantify different gene expression. Multi channel makes use of only one test to give multiple results but different samples sometimes interfere with each other leading to non unique results.
Applications:
(i)Diagnostic applications:
Micro array technology can be used to detect presence of genetic diseases, presence of mutations, polymorphism, cancer etc.
(ii)Gene expression profiling:
Using this technique, the number of genes present, the expression rate each gene can be determined. Monitoring the gene expression is known as gene expression profiling. Quantitative and qualitative measurements of such expressions is possible with the help of microarrays.
(iii)Genomics:
With the help of microarrays, SNPs or single nucleotide polymorphisms can be detected. It refers to different nucleotides present at the same base position in different alleles. Such minute differences between different individuals or different alleles of same gene can be identified successfully and uniquely with the help of micro arrays.
(iv)Sequencing:
A microarray is prepared with oligonucleotides of specific length. When hybridized with target sequences of unknown lengths, the results obtained indicate the length of the target sequence.
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