L5: Polymerase Chain Reaction Flashcards
Go through the history of polymerase chain reaction
1983/93: Nobel Kary Mullis for rapid amplification
What are the requirement for successful PCR
- dsDNA template
- primers (ssDNA, 8-30mers)
- dNTPs
- thermostable DNA polymerase
- Mg2+ buffer
What is a thermocycler
- alters temperature on programmed cycle
What is Taq DNA polymerase
- thermostable DNA polyermase
- active at 70-80, surives at 97 but at low fideltiy
- lower error rate than DNA pol 1
List options for thermostable enzymes
- Pfu
- Vent (high fidelity w/ 3’-5’ exonuclease activity)
Discuss primer specificity
- Typical primers are 8-25 nt
- annealing temps are 2-5ºC below Tm of primer
- if temp is too low, non specific priming can happen
List 5 applications of PCR
- selective amplifciation of DNA pieces
- DNA sequencing
- Site directed mutagensis
- Genomic Mapping
- Quantification
List 9 types of PCR
- Conventional (Qualitative
- Multiplex
- Nested
- RT and qRT
- Quantitative
- Hot Start
- TouchDown
- Colony
- Methylation specific
What is Gel electrophoresis based on , media usage
- separates charged molecules based on net charge carried + size
- Agarose as frictional media of DNA/RNA larger than 250
- Acrylamide as frictional media for high resolution 50-1000
How do you use PCR as molecular marker
- use PCR and 10-21 nt primers to detected polymorphisms
What are microsatellite markers, how are they used
- simple sequence repeats (SSR markers)
- short sequences of v short repeats in tandem arrays with unique euk DNA throughout
- each locus has several alleles => each allele has distinct band
–
how : microsatellitles repeats are found in euk genomes, create primers with short repeats and tes
How do you use PCR to test for hybridity
- do banding profile of parents and progeneis
- true: bands from both parents
- false: bands from single parent
extract DNA => select marker => amplify
What must you bear in mind for PCR amplified transposon flanking sequencing and their annotation
~87% of insertions are either known or putative genes
How do you perform PCR system for gene expression w/ RT
- isolate RNA from tissues of intereset
- eliminate DNA from sample
- make cDNA from mRNA
- do PCR on sample with trasngene specific primers
How do you do PCR for gene expression w/ Q PCR
Q PCR
* use fluorescence as an output for amplification
* the amount of starting template DNA depends on Ct number
==> more DNA means lower Ct number
