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PCR. استاد مربوطه:خانم مهندس رخشی ارائه دهنده: سوسن حسینی. DNA Replication in the Tube PCR. Polymerase Chain Reaction Most important recent discovery (1985) Patented – all PCR reactions pay royalty Repeated replication of specific DNA sections Small quantities Feathers, hair etc.
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PCR استاد مربوطه:خانم مهندس رخشی ارائه دهنده: سوسن حسینی
DNA Replication in the TubePCR • Polymerase Chain Reaction • Most important recent discovery (1985) • Patented – all PCR reactions pay royalty • Repeated replication of specific DNA sections • Small quantities • Feathers, hair etc. • Specific regions of DNA • Target specific sequences • Logarithmic replication • 2 4 8 16 32 64 128 256 512 1028
PCR • How does it work: • Separate the two strands (94oC) • Anneal primers (55oC) • Replication start • Extension (72oC) • = replication • Repeat 20 – 30 times 94° 94° 72° 55°
PCR in practice • Needs accurate temperature control • PCR machines • Automatic cycling of temperature • Reaction ingredients • Buffer • Keep pH constant • Template DNA • Primers • As a starting point • Forward and reverse • Nucleotides • To synthesize DNA • Polymerase • Taq polymerase • MgCl2 • Aids enzyme activity
DNA Replication in the TubePCR • Need PCR primers • Polymerase can only start synthesizing from double stranded DNA • Start where primer anneal • What are primers? • Short artificial DNA sequences • 15-20 bp • Match template DNA • Can pick where we want to start PCR • Which direction?
Action of DNA polymerase is always 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
DNA sequences are always written 5’ 3’ 5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’ 3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’ 5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’ 3’-ACGT-5’ 5’-GCCA-3’ 3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’ So the Primers are 5’-GCCA-3’ 5’-TGCA-3’ and
PCR primers • Annealing temperature • Optimal temperature for primers to attach to the template DNA • Too high • Bonds don’t work • Primer doesn’t anneal • Too low • Primer may attach anywhere • ‘Non-specific amplification’ • Depends on strength of bonds • Remember: • G-C – three hydrogen bonds • A-T – two hydrogen bonds • Annealing temperature dependson GC content
Primers • Where do we get primer sequences from? • Somebody may have isolated them • Check databases • Freely available on internet (GenBank) • Results not publishable without primer information • Heterologous primers • Isolated from related species • Very useful for many applications • Problem • may not exactly match • PCR does not always work • Primer design from published sequences • Align related species • Design primers in conserved regions • Amplify variable regions • Primer isolation • Very lengthy and expensive procedure • several months work
Primer design ATA GGC GCC 5’-ACTGT AGAT-3 • Primer pairs should have similar annealing temp • length, %GC content • Tm = 4(G + C) + 2(A + T) oC. • Primers should have no self complementarity • Minimal (<3bp) between-primer-complementarity 5’-ACTGTGCCATAGATGCAG-3’ |||| 3’-CAACTGCACCGTATGCAT-5’ • Programs on the web to design primers • Links on webpage
PCR - in practice Sample Single Reaction Template DNA 1-2 µg genomic 1-2 µg mtDNA 1µl Forward Primer 10 mM 2.5 µl Reverse Primer 10 mM 2.5 µl dNTPS 8mM 2.5 µl Mg++ 20mM2.5 µl 10X buffer 2.5 µl H2O 11.5 µl Taq 0.5 U >1 µl Total 25 µl Primers, dNTPS and Mg are often made up as 10X stocks for ease of setting up reactions Buffer is polymerase-specific, purchased with the enzyme, Caution: some buffers are Mg++ free, others are not Use high quality nuclease free water
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