1DNA methods summary1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins “expressed”. (N)3. Gel electrophoresis separates DNA on the basis of size.4. DNAs can be synthesized (up to ~100 bases commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain termination. (N)7. Oligonucleotides can be used to change bases by “site-directedmutagenesis”. (N)8. “Southern” blotting detects sequences by hybridization.9. Genes can be knocked out (deleted) or replaced in prokaryotes andeukaryotes. (N)10. Microarrays detect gene expression patterns over the genome.Restriction enzymes cut DNA at specific sites2Restriction enzymes cut DNA at specific sitesPalindromeRestriction enzymes cut DNA at specific sitesPalindromeDoc note, I dissent. A fast never prevents a fatness. I diet on cod.3Restriction enzymes cut DNA at specific sites• 3 types of ends: 5’ overhang, blunt and 3’ overhang• Cognate methyl transferases protect host genome from digestion.Restriction-modification systems degrade “foreign” DNA.Average frequency of restriction sites in“random” DNA sequencesThe average occurrence of each sequence = 1/4n,where n = the site length and all bases are equally representedSite size468Average frequency 1/2561/4,0961/65,536(1/4 x 1/4 x 1/4 x 1/4)4Lots of different recognition sites knownCore four basesFlanking basesNoneA----TC----GG----CT----AA simple cloning procedure1. Cut “insert” and “vector”DNA with a restrictionenzyme2. Mix and join ends withDNA ligase. The endsshould match forefficient ligation.5 Cloning without DNA ligase1. Prepare open vector and insertwith the same long “sticky” ends + Pol I Klenow fragment + dATP2. Mix and let the ends anneal.3. Transform the nicked plasmid.The plasmid is repaired in vivo.1. Prepare an insert flanked bysites for a site-specific DNArecombinase.2. Mix insert with the closedvector containing therecipient recombination siteand recombinase enzyme. +3. (Have lunch.) Transform.Ligation-independent cloning“Gateway” cloningE +No dT in templateTTTTAATAGC~20 base pairsVector InsertVectorInsertInsert Vector“Vectors” allow DNA sequences to be cloned - 1Phage λ for cloningbig (7-25 kb) DNApiecesOri + selectablemarker + cloningsite (polylinker)6“Vectors” allow DNA sequences to be cloned - 2“Reporter” genes:β-gal, GFP . . .Shuttle vectors:move genes betweenorganismsExpression vectors:Make your favorite protein“Vectors” allow DNA sequences to be cloned -3Transient transfection: eukaryotesStable transfectionPlasmid is unstable --Expression variable Plasmid integrated in large tandem arrays -- protein overexressed7Gel electrophoresis separates DNA on the basis ofsizeAgarose: big fragments (>300 bp)Acrylamide: smaller fragments, higher resolutionMobility proportional to log MW.Chemical DNA synthesisSequential rounds ofcoupling, oxidationand deprotection ofthe 5’ OH build upthe oligonucleotide.3’5’8Frontiers in DNA synthesisCurrently: 100-200 nucleotides routine (Assemble 5 kB)10,000 = largest.Primer set for the human genome (30,000 genes) ~ $104Goal 1: Make yeast chromosome 3: 300 kB without errors! (Jeff Boeke; $300,000)Goal 2: Assemble a total of 16 X 106 w/o errors for ~$1000 (George Church)PCR (Polymerase Chain Reaction): isolate andamplify any DNA sequenceN cycles amplifies the target sequence 2N-fold.Quantitative PCR (QPCR) defines amount of starting template.Copies: 1 2 4 89DNA methods summary1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins “expressed”. (N)3. Gel electrophoresis separates DNA on the basis of size.4. DNAs can be synthesized (up to ~100 bases commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain termination. (N)7. Oligonucleotides can be used to change bases by “site-directedmutagenesis”. (N)8. “Southern” blotting detects sequences by hybridization.9. Genes can be knocked out (deleted) or replaced in prokaryotes andeukaryotes. (N)10. Microarrays detect gene expression patterns over the genome.DNA sequencing by partial chain terminationddNTPs terminatethe chain10DNA sequencing by partial chain terminationSmall amount of ddGTP + excessdGTP partially terminates chains atCs in the templateddNTPs terminatethe chainDNA sequencing by partial chain termination1. All fragments start at theprimer2. All fragments ending in aparticular base have adifferent length and adifferent color tag3. Separating the mixture ofproducts by size reveals thesequence.4. <1000 bases/reaction11Two strategies for genome sequencingHierarchical Shotgun Sequencing SequencingGenome resequencing -- Many short reads1. Prepare sample: Shear, repair,ligate adapters to both ends2. Create clusters: Attach one endto a solid support, PCR in situ withone primer attached to supportRandom DNA fragment3. Sequence-by-synthesis:• Denature, add primer + all 4fluorescent dNTPs with blocked 3’OH to add 1 base to each cluster.• Read each cluster with laser.• Deblock 3’ OH and remove color.• Repeat synthesis of next base.• Read.• Repeat 30 x for 106 clusters!4. “Assemble” genomesequence by finding overlapsof 30mers and comparing toknown genome sequence.PCRClusterssDNA surroundedby a lawn of primers12Genome resequencingCurrently:109 reads ~ $5,000 X-Prize:Human genome < $1,000(3.2 x 109 bases (x 8 reads)) Site-directed mutagenesis1. Denature methylatedtemplate and annealdivergent mutagenicprimers.2. PCR amplify the entireplasmid with a DNA pollacking 5’-->3’ exonuclease.3. Select against parentalstrands with Dpn1restriction enzyme, whichcuts methylated andhemimethylated DNA.4. Transform-CH3 PCRDpn1-CH3 CH3-CH3--CH3 -CH3 -CH3 -CH3 CH3-Digested13Gene replacement in mice -- make donor cells1. Insert drug markers intogenome of ES cells2. Select to enrich forhomologous recombinantsCheck insertion site bySouthern blottingNeor confers resistance to G-418.tkHSV confers sensitivity to ganciclovir.“Southern” blotting detects DNA sequences byhybridization1. Digest DNA usingrestriction enzyme(s)2. Run gel3. Transfer DNA from gelto (nitrocellulose) paper.4. Denature DNA, hybridizeprobe DNA, and washoff excess probe.5. Detect the probe on thepaper. E.g. byautoradiography.“Northern” blotting detects RNA on the gel.14Gene replacement in mice -- germline
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