ZFNsTALENsCRISPRsT009a-0yYYe-EEEoFoklFoklCas9MMVTYDouble stranded DNA BreakYYCell can't repair breakVGene demolished9-11
9-11 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
1978Nobel Prize inPhysiology or MedicineDanielNathansWernerArberHamiltonO.SmithFor the discovery of restriction enzymes and their application toproblems of moleculargeneticsBCDEFHIJKAGDigestedSV40DNA9-12
9-12 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 1978 Nobel Prize in Physiology or Medicine For the discovery of restriction enzymes and their application to problems of molecular genetics. Digested SV40 DNA
Different restriction enzymes producefragments ofdifferentlengths(a)CalculatingAverageRestrictionFragmentSizeProbability that a1.Probabilitythatafour-baserecognitionsitewill befoundinagenome=four-base1/4×1/4X1/4×1/4=1/2562.Probability thata six-base recognitionsitewill be found recognition site will1/4×1/4×1/4×1/4×1/4×1/4=1/4096be found in the(b)RestrictionSitesinHumanGenomicDNAgenome=1.Four-base1/ × × / × % = 1/2561kbRsal sites2.Six-baseEcoRIsites3.Eight-baseNotl sites9-13Fig.9.3
9-13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Different restriction enzymes produce Different restriction enzymes produce fragments of different lengths fragments of different lengths Probability that a Probability that a four-base recognition site will recognition site will be found in the be found in the genome = genome = ¼ ¼ ¼ ¼ = 1/256. = 1/256. Fig. 9.3
Different restriction enzymes produce differentnumbers offragmentsRsal (4bpenzyme)3,000,000,000bp/256bp=~12,000,000fragmentsEcoRI (6bpenzyme)3,000,000,000bp/4096bp=~700,000fragmentsNotI (8 bp enzyme)3,000,000,000bp/65536bp=~46,000fragments9-14
9-14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Different restriction enzymes produce different Different restriction enzymes produce different numbers of fragments numbers of fragments RsaI (4 bp enzyme) enzyme) 3,000,000,000 3,000,000,000 bp / 256 bp = ~ 12,000,000 fragments = ~ 12,000,000 fragments EcoRI (6 bp enzyme) enzyme) 3,000,000,000 3,000,000,000 bp / 4096 bp = ~ 700,000 fragments = ~ 700,000 fragments NotI (8 bp enzyme) enzyme) 3,000,000,000 3,000,000,000 bp / 65536 / 65536 bp = ~ 46,000 fragments = ~ 46,000 fragments
Partial digests are used so enzymes cut only a subset of thetotal number of recognition sitesin agenome(b1(a)EcoR1SitesinHumanGenomicDNAOnerandomEcoR1site out of five on eachmoleculeiscutbyenzyme↓+?10 1 10 1 11 101 1 1 01110++↓+EcoR111 0 11 1 1 01 111 0 1 111011sites9-15
9-15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Partial digests are used so enzymes cut only a subset of the Partial digests are used so enzymes cut only a subset of the total number of recognition sites in a genome total number of recognition sites in a genome