True about RFLP: (PGI Dec 2006)

Ans: A (Detects mutation); B (Recognizes tribnucleotide repeats); C (Detects deletion) & D (Blunt ends produced) Restriction Fragment Length Polymorphism (RFLP)# RFLP is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes'' and analyzing the size of the resulting fragments by gel electrophoresisQ.# Restriction Endonuclease (RE) cleaves ds-DNA into smaller manageable fragments, at specific sites. RE can produce 'sticky' and 'blunt1 ends.# Analysis of RFLP variation is an important tool in genomic mapping, localization of genetic disease, determination of risk for a disease, genetic fingerprinting, and paternity testing.# It is the sequence that makes DNA different, obtained from different sources, and RFLP analysis is a technique that can identify differences in the sequence (when they occur in a restriction site).# The basic technique for detecting RFLPs involves fragmenting a sample of DNA by restriction enzymes, which can recognize and cut DNA wherever a specific short sequence occurs, by a process known as a restriction digest. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to membrane via the Southern blot procedure. Hybridization of the membrane to a labelled DNA probe then determines the size of the fragments which are complementary to the probe. An RFLP occurs when the size of a detected fragment varies between individuals. Each fragment size is considered an allele, and can be used in genetic analysis.Table (Harrison 17th/406): Methods Used for the Detection of MutationsMathodPrincipleType of Mutation DetextedCommonly Used TechniquesCytogeneticanalysisUnique visual appearance of various chromosomesNumerical or structural abnormalities in chromosomesFluorescent in situ hybridization (FISH)Hybridization to chromosomes with fluonescently labeled probesNumerical or structural abnormalities in chromosomesSouthern blotHybridization with genomic probe or cDNA probe after digestion of high molecular DNALarge deletion, insertion, rearrangement, expansions of triplet repeat, amplification MathodPrincipleType of Mutation DetctedCommonly Used TechniquesPolymerase chain reaction (PCR)Amplification of DNAsegmentExpansion of triplet repeats, variable number of tandem repeats (VNTR), gene rearrangements, translocations; prepare DNA for other mutation methodsReverse transcriptase PCR (RT-PCR)QReverse transcription, amplification of DNA segment - absence or reduction of mRNA transcriptionAnalyze expressed mRNA (cDNA) sequence; detect loss of expressionDNA sequencingDirect sequencing of PCR productsPoint mutations, small deletions and insertions Sequencing of DNA segments cloned into plasmid vectors Restriction fragment polymorphism [RFLP) QDetection of altered restriction pattern of genomic DNA (Southern blot) or PCR productsPoint mutations, small deletions and inser - tionsQ