PCR Flashcards
How is the temperature chosen?
Based on what primers are for annealing stage. Inbetween 55-60. Also at amplification stage different polymerases function better at different temperatures.
What are the reaction components for a PCR reaction?
Target DNA, pair of primers (oligonucleotides) define sequence to be amplified, dNTPs (DNA building blocks), Mg++ ions (cofactor of the enzyme), buffer solution maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme.
What is the effect of water as a PCR component?
Purity, contamination, needs to be nuclease free to not get false negatives and positives when diagnosis people with disease.
What is in the buffer?
Must match polymerase, store with polymerase frozen, comes as a concentrate, typically contain KCl and Tris (specific to polymerase) may contain DTT (reducing agent).
What are the factors to consider with the DNA template?
Amount of DNA present. Less= more cycles, complexity of DNA e.g., plasmid vs whole genome, purifying (interfering factors like enzymes), degredation, contamination, presence of poisons e.g., EDTA which scavengers Mg++
What do you have to do with magnesium and DNA?
Add more magnesium than EDTA so enough magnesium left for polymerase.
What are some examples of polymerases?
Used to use Taq. Now use Q5 High fidelity DNA polymerase, Q5U hot start high fidelity DNA polymerase. Factors to consider are: Cost, proofreading, error rate, hot start, ends of products.
What do primers need to start with?
3’ OH which allows you to direct where the polymerase starts amplifying.
What does a quick change reaction allow?
User to introduce a mutation.
What are random hexamers?
Short oligonucleotides with random base sequences usually [d(N)6]. Used to prime mRNAs with or without poly(A) for cDNA synthesis.
What are oligo dT primers?
String of 12-20 deoxythymidines that anneal to poly(A) tails of eukaryotic mRNAs.
What is HPLC?
High performance liquid chromatography. Purification over a column. Just need desalting and maybe this for standard PCR. In special cases, PAGE.
What factors need to be considered when trouble shooting primers?
Age, number of freeze thaws, contamination, sequence, amount can vary over a wide range 100-500nM is typical. Too low/too high gives low amplification.
What to consider with nucleotides?
200-400 micromolar works well. Too much leads to mispriming and errors and can scavenge Mg++. Too low gives faint products. Age number of freeze thaws (just 3-5 cycles) dilute in buffer to prevent acid hydrolysis, contamination.
What amount of Mg++ is suggested?
0.5-3.5 micro molar. Too low Taq won’t work. Too high mispriming.
What factors to consider with DNA polymerase?
Thermostable (activity declines with time at 95 degrees), matches buffer? age, contamination, storage (kept on ice), concentration typically 0.5 to 1 units per reaction. Stored in glycerol so always liquid when frozen so no need to thaw but sometimes not mix due to this.
What extras can be added to PCR?
Proprietary, glycerol or DMSO (stabilize Taq, decrease secondary structure. May help or hurt depending on primers. Typically already in the Taq stock.) BSA frequently helps, doesn’t hurt. Betaine useful for GC rich templates.
What is quantitative PCR?
Used with reverse transcription (e.g., to measure the level of mRNA gene expression) assay uses a standard curve to quantitate the amount of target present using a fluorescence-labelled probe for detection. Each technique uses some kind fluorescent marker which binds to the DNA.
What are reverse transcriptases for?
Forming cDNA libraries. Contain DNA copies of mRNA from cells tissues.
What are Taqman probes?
Fluorescent labelled oligonucleotides (TaqMan probes) Taqman probes are complementary to a region of the target gene. The 5’ to 3’ exonuclease activity of the polymerase cleaves the probe, releasing the fluorophore into solution.
What is SYBR green I?
A fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to dsDNA products. Binds to the minor groove of the DNA double helix with a higher affinity for dsDNA that for ssDNA. Cost effective. Not sequence specific.
What are the advantages of TaqMan?
Increased specificity. Use when the most accurate quantitation of PCR product accumulation is desired. Option of detecting multiple genes in the same well.
What are the disadvantages TaqMan?
Relative high cost of labeled probe.
What are the advantages SYBR Green I?
Relative low cost of primers. No fluorescent labeled probes required.
