MethodsforAnalysisofDNANucleic acid hybridization and probesSingle-stranded DNA can unite with other single-strandedDNA or RNA, and RNA can unite with other RNA -hybridizationFoundation for gene probes-short DNA fragments of aknown sequence that will base-pair with a stretch of DNAwith a complementary sequence, if one exists in thesampleUseful in detecting specific nucleotide sequences inunknownsamples- Southern blot method - DNA fragments areseparated by electrophoresis, denatured, andthenincubated with DNA probes. Probes will attach to acomplementary segment if present.-Isolatefragmentsfromamixoffragmentsandfind16specific gene sequences
16 Methods for Analysis of DNA • Nucleic acid hybridization and probes • Single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can unite with other RNA – hybridization • Foundation for gene probes – short DNA fragments of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample • Useful in detecting specific nucleotide sequences in unknown samples – Southern blot method – DNA fragments are separated by electrophoresis, denatured, and then incubated with DNA probes. Probes will attach to a complementary segment if present. – Isolate fragments from a mix of fragments and find specific gene sequences
ICDNA Probe (DNA Hybridization)GrawHHi品0EPlayPauseAudioTextAthightemperatures,double-strandedDNA willdenature or separate into singlestrands.When the temperature is lowered,the two strands will anneal becauseofthebase pairinginteractionsof the complementary strands.CopyrightTheMcGraw-Hill Companies,Inc
FEXTheSouthernBlotCopyright @The McGraw-Hill Companies, Inc.Permission required for reproduction or display231DNAsamples are cut withrestriction enzy mes and loadedon agarose gel for electrophoresis.Lane 1: Labeled size markersLane 2: DNA cut with restriction enzy me ALane 3: DNA with restriction enzy me BGel electrophoresis3WeightDNA is denatured;Paper towels gel is placed on-DNA-binding fitersponge wick.一GelWick (sponge)一BufferDNAis separatedby2electrophoresis and visualized byDNA-binding filter,paper towels,andstaining and photography in UV light.weight are placed on gel. Buffer(When DNAtravels, it appears as asmear rather than distinct bands.)passes upward by capillary actiontransferringDNAfragmentstofilterFilter is placed in heat-sealed food bagwithsolutioncontainingradioactiveprobe.23Overlay filterDevelopedX-rayfilmwith X-ray filmwith DNAbands18Filteris washed toremoveexcess probe,thendried. Film is then exposed to produce photographicimage of DNA bands that reacted with the probes
18 The Southern Blot DNA samples are cut with restriction enzy mes and loaded on agarose gel f or electrophoresis. Lane 1: Labeled size markers Lane 2: DNA cut with restriction enzy me A Lane 3: DNA with restriction enzy me B 1 2 3 Gel electrophoresis DNA is denatured; gel is placed on sponge wick. Weight Paper towels DNA-binding f ilter Gel Wick (sponge) Buf f er DNA is separated by electrophoresis and v isualized by staining and photography in UV light. (When DNA trav els, it appears as a smear rather than distinct bands.) DNA-binding f ilter, paper towels, and weight are placed on gel. Buf f er passes upward by capillary action transf erring DNA f ragments to f ilter. Filter is placed in heat-sealed f ood bag with solution containing radioactiv e probe. Filter is washed to remov e excess probe, then dried. Film is then exposed to produce photographic image of DNA bands that reacted with the probes. Ov erlay f ilter with X-ray f ilm Dev eloped X-ray f ilm with DNA bands 1 2 3 1 2 3 1 2 4 5 3 Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
MCSouthern BlotHirawOrganismAOrganismBOrganismC宁布烫品国PlayPauseAudioTextThe Southern blotis used to verifythe presenceorabsence ofa specificnucleotide sequence in the DNA from different sources and to identify the size oftherestrictionfragmentthatcontainsthesequenceCopyright@TheMcGraw-Hill Companies,Inc