MBIO 3410 lecture Nov 1 2007

MBIO 3410 lecture Nov 1 2007 - Last Lecture Overview...

Info iconThis preview shows pages 1–8. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Last Lecture Overview Artificial chromosomes (continued) PACs ( P1 artificial chromosome ) Cloning with P1 vectors Advantages of the P1 DNA packaging method Bacterial artificial chromosomes ( BACs ) BAC pros and cons Human artificial chromosomes ( HACs ) Transfection of eukaryotic cells Shuttle vectors Yeast episomal plasmids Agrobacterium tumefaciens Ti plasmid Baculovirus Mammalian viral vectors
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
2 PCR
Background image of page 2
3 Polymerase chain reaction (PCR) Key concepts PCR is the amplification of specific DNA sequences in vitro PCR requires: Two primers - one that is complementary to each strand of DNA DNA polymerase Repetitive heating and cooling cycles amplify the DNA between the two primer binding sites to yield large quantities of replicated DNA
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
4 PCR PCR involves two oligonucleotide primers usually between 17 and 30 nucleotides in length which flank the DNA target sequence that is to be copied One of the primers is the same sequence as one strand of the DNA (sense strand) while the other primer is the same sequence as the other DNA strand (antisense strand) The sense strand primer will bind through complementary base pairing interactions to the antisense strand and will initiate DNA synthesis of a new sense strand Similarly, the antisense primer will bind to the sense strand of the DNA and will initiate the synthesis of a new antisense strand The PCR reaction is split into three separate stages each of which is performed at a different temperature The cycle of denaturation–annealing–extension is repeated 20–30 times in order to achieve satisfactory amplification of a specific DNA sequence
Background image of page 4
5 Denaturation The two strands of the target DNA molecule are separated into two strands by heating This step is most often performed at 94 C for ~60 seconds The denaturing and annealing steps are relatively short, but are sufficient to allow breaking and reforming of the hydrogen bonds between DNA strands
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
6 Annealing The two target strands are then allowed to cool in the presence of the oligonucleotide primers One of the primers recognizes and binds to one of the target DNA strands and the other primer recognizes and binds to the other strand The primers are designed such that the free 3’-end of each primer faces the other one and so DNA synthesis proceeds on both strands through the region between the two primers The temperature at which annealing of the primers to the template DNA occurs depends upon the length and sequence of the primer and the level of specificity required in a particular PCR reaction Typically a temperature in the range of 45-60 C would be chosen for ~30 seconds
Background image of page 6
7 Extension A DNA polymerase binds to the free 3’-end of each of the bound oligonucleotides and uses dNTPs to synthesize a new DNA strand in a 5’- to 3’-direction
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 8
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 07/05/2008 for the course MBIO 3410 taught by Professor Richardson during the Fall '07 term at Manitoba.

Page1 / 28

MBIO 3410 lecture Nov 1 2007 - Last Lecture Overview...

This preview shows document pages 1 - 8. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online