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#DNA fingerprinting

4 public questions tagged with this topic.

RAPD markers are generally:

Random Amplified Polymorphic DNA employs single decamer arbitrary primers to amplify random genomic segments without prior sequence information using low stringency PCR annealing at 36°C. Amplified fragments present as bands indicate primer binding sites present in both orientations within amplifiable distance; absence indicates sequence divergence or insertion preventing amplification. Since technique detects presence versus absence of dominant allele but cannot distinguish heterozygous Aa from homozygous dominant AA, both produce band, it behaves as dominant marker with 3:1 segregation. Moreover, RAPD is sensitive to template quality, primer concentration, MgCl2, Taq polymerase brand, and thermal cycler profile, causing poor reproducibility between labs and faint artifact bands and inconsistent results. Despite advantages of low cost, quick assay, requirement of nanograms of DNA and no prerequisite genomic library, modern breeding has shifted to more robust codominant SSR and SNP markers because RAPD repeatability issues compromise mapping and selection reliability and publication standards. ISSR, AFLP, and SRAP markers share similar limitations as dominant markers, but development of sequence characterized amplified region SCAR markers from RAPD fragments improves reproducibility by converting random fragment into locus specific codominant PCR assay, bridging low cost random approach with reliable sequence based diagnostics for breeding.

Ref: Williams JGK et al. 1990 Nucleic Acids Res – RAPD dominant reproducible issues; Welsh & McClelland 1990

Which statement is correct about AFLP?

Amplified fragment length polymorphism involves sequential steps of complete genomic DNA digestion with rare and frequent cutting enzymes, ligation of double-stranded adapters to fragment ends, pre-selective amplification with primers having one selective nucleotide, followed by selective amplification with primers containing two or three additional selective nucleotides labeled for detection. This two-step PCR reduces complexity and increases specificity, generating fifty to one hundred scorable bands per reaction. Random primer methods or single-step protocols lack this nested selectivity and reproducibility characteristic of AFLP fingerprinting.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

The most polymorphic marker system is:

Simple sequence repeats exhibit exceptional polymorphism due to high mutation rates caused by slipped-strand mispairing during DNA replication. Variable repeat numbers generate many alleles per locus, often exceeding ten in diverse germplasm, providing high polymorphic information content and heterozygosity values. Their codominant, multiallelic nature surpasses biallelic SNPs, low polymorphism RFLPs and dominant RAPD markers that detect fewer loci. Widespread genomic distribution, excellent reproducibility and ease of PCR detection further enhance utility for linkage mapping, population structure and fingerprinting studies in crop plants.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.