Q.26 Among individuals in a human population, minor variations exist in nucleotide
sequences of chromosomes. These variations can lead to gain or loss of sites for
specific restriction enzymes. Which of the following technique is used to identify
such variations?
(A) Polymerase dependent fragment insertion
(B) Real-time polymerase chain reaction
(C) Restriction fragment length polymorphism
(D) Reverse transcriptase polymerase chain reaction
Restriction Fragment Length Polymorphism (RFLP) is the technique used to identify minor nucleotide sequence variations in human populations that create or eliminate restriction enzyme sites. These variations result in DNA fragments of different lengths after enzyme digestion, detectable via gel electrophoresis. The correct answer is option (C) Restriction Fragment Length Polymorphism.
Correct Answer
Option (C) Restriction fragment length polymorphism precisely matches the query. RFLP exploits polymorphisms where single nucleotide changes alter restriction sites, producing distinguishable fragment patterns. This method analyzes genomic DNA digested by restriction enzymes, separated by size on agarose gels, and often visualized via Southern blotting.
Option Explanations
Each option relates to molecular biology but differs in purpose:
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(A) Polymerase dependent fragment insertion: Refers to PCR-based cloning techniques where polymerases like Pfu generate blunt-end fragments later modified (e.g., A-tailed) for vector insertion. No role in detecting restriction site variations.
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(B) Real-time polymerase chain reaction: Quantifies DNA/RNA via fluorescence during amplification cycles, ideal for expression analysis or copy number but ignores fragment length differences from restriction digests.
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(C) Restriction fragment length polymorphism: Directly detects site gains/losses via enzyme-specific cleavage, yielding variable fragment lengths separated electrophoretically (PCR-RFLP variant amplifies first). Core method for such polymorphisms.
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(D) Reverse transcriptase polymerase chain reaction: Converts RNA to cDNA for amplification, used in gene expression studies, not genomic DNA variations or restriction patterns.
Applications in Genetics
RFLP revolutionized early genotyping, mapping disease loci like Huntington’s, though largely replaced by PCR and sequencing for higher throughput. In research, it confirms polymorphisms in population studies or forensics via banding patterns. For biotech students in Jaipur or Rajasthan exams, mastering RFLP links nucleotide diversity to practical assays.
