1
Who invented PCR and when?
Kary Mullis in 1986
Describe the 4 things that make PCR the ideal diagnostic test.
1) Specificity- yields positive result for target molecule only
2) Sensitivity- detects small amount of antigen
3) Simplicity- runs efficiently and inexpensively on a routine basis
4) Speed- finishes in shortest time possible
What is sensitivity? How is it calculated?
The ability to identify correctly affected individuals
=true positives/affected individuals
What is specificity? How is it calculated?
The ability to identify correctly non-affected individuals?
=true negatives/non-affected individuals
What is complimentary hybridization?
Describes how the strands of DNA are complimentary to one another (G always goes with C with 3 H bonds, A always goes with T with 2 H bonds)
What are the components of PCR?
Template DNA, primers, dNTPs, heat-stable DNA polymerase (Taq polymerase), buffer
Describe the 3 step thermal cycling involved in PCR.
1) Heat denaturation- 95 C
2) Primer annealing- 55 C
3) Primer extension/elongation- 72 C
What is a primer?
A small oligonucleotide with complementary sequence to the target DNA molecule
Why is a heat-stable DNA polymerase necessary for efficient PCR?
It must be able to withstand the drastic temperature changes for thermal cycling
What is gel electrophoresis?
What is included in classical PCR vs. real time PCR (qPCR)?
Classical PCR- primers for amplification, no probes for detection
Real Time PCR- primers for amplification, probes for detection
What are SYBR green and FRET examples of?
Molecules that cause fluorescence for visualization of DNA
What are the advantages of Real Time PCR over Classical PCR?
Real Time PCR is less prone to carry-over contamination, can be quantitative (as opposed to just qualitative), can differentiate via melting curve analysis and is more robust than Classical PCR
What is the relationship between melting temperature and the match between the probe and amplicon in Real Time PCR?
A high Tm indicates a full mismatch, a low Tm indicates a mismatch
How do you distinguish if the DNA is homozygous wild type, homozygous mutant or heterozygous?
If the peak of the graph is heavily on 1 side, it is homozygous and it can be determined as either wild type or mutant based off the control, if the curve is bimodal it is most likely heterozygous
Which has a higher melting point: Hepatozoon americanum or Hepatozoon canis? What does this say about the 2?
Hepatozoon americanum has a higher melting point (more G:C) than Hepatozoon canis so Hepatozoon americanum is determined to be more dangerous than Hepatozoon canis
What is RT-PCR? What is qPCR?
RT-PCR is reverse transcription PCR
qPCR is quantitative (or real-time PCR)
They are NOT the same thing
What are the different ways DNA can be sequenced?
Whole RNA Seq with ribosomal depletion, mRNA Seq with poly A selection, sequencing of other RNA species, nuclear RNA Seq
How is Next Gen Sequencing classified?
By the starting material: DNA Seq vs. RNA Seq
What is Next Gen Sequencing?
DNA fragments are bound to selected protein to analyze the sequences of DNA binding sites of the protein of interest or localization of histone modifications (ChIP Seq)
Why is detection of the amplification product in qPCR with probes more specific than with double-stranded DNA binding fluorescent dyes?
Fluorescent probes target DNA with complementary sequences while intercalating dyes cannot distinguish target DNA products from other double-stranded DNA products present in the sample