DNA markers are used extensively in genetic mapping studies. They are fragments of DNA which exhibit polymorphism. Genetic mapping using DNA markers are used to identify chromosomal location of mutant genes involved in genetic diseases.
Different types of markers are in use.
a.Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism was the first method used to generate markers for genetic mapping. The gene locations are identified in relation to these linked markers.
Genomic DNA is digested with restriction enzymes which results in restriction fragments. These fragments are separated using agarose gel electrophoresis. These DNA fragments are denatured using an alkali which provides single strand (ss)DNA fragments. These ss DNA fragments are blotted onto nitrocellulose filter paper. The different fragments are identified using the presence/ absence of restriction sites and arranged using their fragment size.
Differences in length of DNA fragments are due to the presence or absence of the restriciton sites in DNA. These are cumulatively called as restriction fragment length
RFLPs can result due to mutations or short tandem repeats.
• RFLP markers are co dominant. Hence identifications of patterns of genomes in homozygous and heterozygous individuals are easier.
• May require large amounts of DNA samples since distinct RFLP markers require different restriction enzymes.
b. Cleaved Amplified Polymorphic Sequences (CAPS)
The principle is similar to RFLP procedure. Here, PCR amplification is used in lieu of DNA blot hybridization. The PCR is used to specifically amplify particular regions of the genomic DNA. Selection is achieved by use of specific primers. The amplified fragments are then digested with restriction endonucleases which reveal the DNA polymorphism.
• CAPS markers are codominant in nature allowing identification of homozygous and heterozygous patterns with ease.
• Require only smaller amounts of DNA since PCR amplification is employed.
• Can be easily assayed with agarose gel electrophoresis.
• The procedure requires use of specific primers and hence the sequence must already be known.
Derived cleaved amplified polymorphic sequence (dCAPS)
The method allows generating Pcr bsed markers for known point mutations. It is useful at sites where a point mutation does not alter the restriction stie.
A primer with mismatches and the point mutation is designed which will create a unique restriction site in one of the alleles. Another primer without these mismatches are used to selectively amplify the region. The resultant PCR products are digested with restriction endonucleases and assayed.
c. Random Amplified Polymorphic DNA (RAPD)
RAPD markers are based on amplification of random DNA fragments with primers. Short oligonucleotide sequences of length app 10 bp are used to hybridize along with the DNA fragments at random at low annealing temperatures.
The fragments in which the oligonucleotide gets hybridized to both DNA strands with sufficient distance in between are selectively amplified. Any changes within this region prevent oligonucleotide binding and essentially amplification. Hence, those fragments with the complementary sequences to oligonucleotide primers get amplified. This results in selective amplification of DNA sequences with RAPD markers.
• Can use random primer sequences.
• RAPD markers are dominant. Hence distinguishing homozygous and heterozygous individuals is not possible.
• Reproducibility of the experiment is very low since low annealing temperatures are used.
• PCR conditions greatly affect the selective amplification of polymorphic bands.
d. Amplified Restriction Fragment Length Polymorphism (AFLP)
AFLP employs two different restriction enzymes, a frequent cutter and a rare cutter. Double strand DNA segments as adaptors are attached to the ends of such restriction fragments and are amplified using PCR. Primers are used which will anneal to adaptor sequences as well as restriction fragments. Either of these primers is labeled and the fragments are separated using electrophoresis.
The 3'ends of the primers have additional nucleotides to limit the amplification. The amplified sequences are compared to identify the polymorphic loci.
• They are typically dominant markers which are unable to provide details on homozygous and heterozygous nature of the gene.
e. Simple Sequence Repeats (SSR) : Microsatellites
Microsatellites are short sequences of DNA repeated in the genome. These motifs are tandem repeats and are usually polymorphic in nature because of the variations in the repeat units. These are called Short sequence repeats or SSR.
Specific primers complementary to the regions of microsatellites are used to identify the SSRs. The hybridization depends on the number of repeats and so does the amplified fragment size. Agarose or polyacrylamide gel electrophoresis is used to separate these polymorphic fragments.
• Does not employ restriction enzymes and avoid the problems such as partial digestion.
• They are co dominant in nature.
f. Single Nucleotide Polymorphisms (SNPs)
SNPs are the commonest forms of polymorphism. They can be detected by using specific primers to amplify the alleles of interest. These are used to identify allele specific polymorphisms.
Identification of SNPs are possible using
1. SSCP- Single strand conformation polymorphism. This is based on difference in patterns of DNA folding due to single nucleotide changes. The mobility of such DNA strands differs in the non denaturing gel electrophoresis.
2. Hetroduplex analysis is doen by identifying the patterns of mobility of homo and heteroduplexed in non denaturing gel electrophoresis or HPLC.
• It is possible to identify mutations due to single base substations which are otherwise non identifiable by conventional mapping.
g. Sequence tagged site(STS)
An STS marker is a DNA sequence found once per haploid set of genome which can be amplified by PCR technique. Hence it is unique to the genome and therefore can map genomes efficiently. STS markers can efficiently incorporate different information to create a physical map. These markers represent coding regions and are therefore widely used in genetic researches.
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