lecture 15 - PCR/next gen sequencing Flashcards
1
Q
what does PCR do
A
amplifies specific sequences of DNA in vitro
2
Q
how does PCR work
A
- pair of oligonucleotides is used to prime enzymatic synthesis of both strands of target sequence
- new duplexes are denatured
- second cycle of synthesis occurs
3
Q
what are the components of PCR mixture
A
oligonucleotide primers, template dna, all 4 dNTPs, heat stable DNA polymerase, buffer
4
Q
what are some pros to pcr over other sequencing mrthods
A
- faster
- more amendable to automation
5
Q
what are some pcr limitations
A
- necessary to know sequences at each end of target DNA so specific primers can be designed
- can be difficult to amplify very long sequences (>15kb)
- some heat stable dna polymerases like Taq don’t proof read and misincoporate bases
4. high sensitivity means that dna contaminiation can be promblem
6
Q
what is quantative pcr and what is it used for
A
version of pcr that includes a fluorescent dye that allows you to determine how much dna was initially present in sample
7
Q
what is reverse transcriptase pcr used for
A
detecting RNA sequences
