CHAPTER 2 (VARIANTS OF PCR) Flashcards
(17 cards)
def of multiplex PCR
amplification of multiple targets in a single PCR reaction using multiple sets of primers to produce amplicons with varying sizes
use to detect more than one organism or to detect multiple genes in one
organism
why multiplex PCR can be tricky?
- Annealing temperatures for each primer: must be optimized to work correctly within a single reaction
- Amplicon sizes: different enough to form distinct bands when visualized by gel electrophoresis.
def of Hot Start PCR
a technique that reduces non-specific annealing of primers and primer-dimer formation. Increase the specificity of PCR by inhibition of polymerase activity at lower temperature
how to achieve inhibition of polymerase activity at lower temperature in hotstart pcr
- cheapest method - manually heating the reaction components to denaturation temp (95C) before adding polymerase
- using antibody interaction
- use wax beads containing Mg++ that can only be released at high temp.
explain usage of antibody interaction to achieve polymerase inhibition
specific antibodies used to block Taq-polymerase at annealing temperature. When the temperature raises to >70 degrees, the specific antibody detaches from Taq polymerase and allows amplification
what does the touchdown pcr used for
use to increase PCR specificity and minimize non-specific PCR products
when does the touchdown pcr useful?
useful if your primers are not 100% complementary to your template DNA (e.g.
degenerate oligos), or when there are multiple members of the gene family you are
amplifying
whats RT pcr or Quantitative pcr
pcr to amplify and quantify targeted DNA
molecule
in RT pcr, amplified DNA is detected as the reaction
progresses in real time.
RT pcr is a ___ system
close
how does the detection/quantification in RT pcr is done
- non specific detection - using no specific dyes like SYBRO green
- specific detection
explain specific detection in RT pcr
- During the PCR annealing step, the probe sticks to your target DNA.
- During the extension step, DNA polymerase moves along the DNA to make a new strand.
- When it reaches the probe, the polymerase chops (degrades) the probe (because polymerase has 5’→3’ exonuclease activity).
- Chopping separates the fluorescent dye from the quencher → 🔥 Fluorescence is now free to glow!
- The more DNA template you have, the more probes are chopped, the stronger the fluorescence detected.
Summary: ✅ Probe binds → ✅ Polymerase chops it → ✅ Dye gets free → ✅ Light comes out → ✅ More DNA = More light!
whats the functions of reverse transcription pcr
- to detect RNA
- detect RNA expression levels
- qualitative study of gene expression.
- detect gene expression through creation
of complementary DNA (cDNA) transcripts from RNA - quantification of RNA, by incorporating qPCR into the technique RT-PCR or qPCR
def of reverse transcription
The transfer of genetic information from RNA to DNA By the enzyme reverse
transcriptase found in retrovirus
whats inverse pcr?
For amplifying anonymous flanking genomic DNA regions - Segments that lie outside from the primers region without any information
whats the application of inverse pcr
- Mapping unknown genomic regions adjacent to known sequences
- Studying structural variations and mutations in DNA
- Cloning unknown sequences next to known genes
- Investigating transposon insertion sites
- Identifying transgene insertion sites in GMOs
- Gene function and regulatory studies
how to avoid cross-contamination by DNA from sources other than the template added
- Work in a laminar flow hood (decontaminate using UV light 254 nm)
- Use PCR dedicated pipettors (with barrier tips), PCR dedicated reagents
- Centrifuge tubes before opening them to prevent spattering, pipet contamination
