Cloning PCR products

Taq polymerase

– usually produces products with single 3' A extension

– can be converted to blunt by T4 DNA polymerase

– blunt end cloning (inefficient)

– or add linker/adaptor for efficient cloning

– can clone in T vectors

– vectors with single 3' T extension

– e.g. cut special vector with XcmI

– (single 3' extension in 9 bp N sequence)

CCANNNNT NNNNTGGCCANNNNA NNNNTGG

GGTNNNN ANNNNACCGGTNNNN TNNNNACC

– T vectors also available commercially

– in PCR cloning kits

– 50 X more efficient than blunt-end cloning

proofreading polymerases – produce blunt ends

– blunt clone or use linker/adaptor

creating restriction sites in primers (any polymerase)

– 3' end of primer is gene-specific

– 5' end of primer is restriction site plus a bit extra

– (most REs do not cleave at end of DNA molecules)

– after PCR, cleave with RE & clone

– no linker needed

– can put different RE sites in 2 primers

– directional cloning

problems:

same RE site within PCR product sequence

– watch for cloned product shorter than original fragment

– use rare-cutter (e.g. NotI) in primer

survival of Taq polymerase in reaction (after RE digest)

– often survives phenol/chloroform

– will fill in 3' recessed end if dNTPs still present

– purify PCR product on gel or ethanol precipitate

– removes enzyme & dNTPs

variations on PCR

inverse PCR (Fig. 6.3)

– PCR out from 2 primers at ends of known sequence

– amplify flanking sequences

protocol:

– digest genomic DNA with RE

– ligate at low DNA concentration

– mostly self-ligate into circles

– PCR with primers for specific sequence

– amplify rest of circle containing specific sequence

– usually several kb

– need proofreading enzyme or mix

uses:

1. probes for chromosome walking

2. finding location of transposon inserts

– identify transposon mutants with interesting phenotype

– do inverse PCR with primers for transposon ends

– use product to probe genomic library

– find original gene transposon inserted in

3. PCR mutagenesis

PCR mutagenesis

– use PCR to produce mutated genetic sequence

– by including mismatch in primer

– inverse PCR out from 2 adjacent primers (with mismatch)

– at mutation site in cloned gene in plasmid

– generates blunt-ended product – ligate & transform cells

anchored PCR

– one gene-specific primer, one vector specific

– can use to identify specific clones in library

– often faster than plaque lifts (& no radiation)

nested PCR – PCR twice, with second primers inside first – controls for false positives (unlikely to happen twice)

reverse transcriptase-PCR (RT-PCR)

– amplification of cDNA by PCR – e.g. RACE

– make first-strand cDNA with reverse transcriptase

– amplify desired cDNA with PCR & gene-specific primer

quantitative PCR

– use PCR to find amounts of specific nucleic acids present

– more template, see PCR product faster

– e.g. find how much of specific mRNA is present

differential display-PCR (DD-PCR)

– use PCR to survey mRNAs produced in different cells

– e.g. cancer cells vs. normal cells

– use poly-T+2N primers for 1^st strand cDNA

– start all strands at 3' ends of mRNAs

– junction with poly-A tail

– PCR amplified with 1^st strand primers (3' end of mRNAs)

- & random mix of 10 nucleotide primers (other end)

– each mRNA amplified as a different-sized cDNA product

– run on gel to sort by size

– look for differences in pattern

– can use these cDNAs as probes to find genes of interest

diagnostic uses

– identify pathogens

– using species-specific primers

– identify chromosome rearrangements (see handout in library)

– associated with some cancers & genetic disorders