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
What is ct number
cycle number when threshold amount od DNA is produced during PCR
What did they find out in barley bvs matl
TLP8 differentially Expressed in Malt and
Feed Barley Varieties
