PCR

Question 1

How is the temperature chosen?

Answer 1

Based on what primers are for annealing stage. Inbetween 55-60. Also at amplification stage different polymerases function better at different temperatures.

Question 2

What are the reaction components for a PCR reaction?

Answer 2

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.

Question 3

What is the effect of water as a PCR component?

Answer 3

Purity, contamination, needs to be nuclease free to not get false negatives and positives when diagnosis people with disease.

Question 4

What is in the buffer?

Answer 4

Must match polymerase, store with polymerase frozen, comes as a concentrate, typically contain KCl and Tris (specific to polymerase) may contain DTT (reducing agent).

Question 5

What are the factors to consider with the DNA template?

Answer 5

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++

Question 6

What do you have to do with magnesium and DNA?

Answer 6

Add more magnesium than EDTA so enough magnesium left for polymerase.

Question 7

What are some examples of polymerases?

Answer 7

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.

Question 8

What do primers need to start with?

Answer 8

3’ OH which allows you to direct where the polymerase starts amplifying.

Question 9

What does a quick change reaction allow?

Answer 9

User to introduce a mutation.

Question 10

What are random hexamers?

Answer 10

Short oligonucleotides with random base sequences usually [d(N)6]. Used to prime mRNAs with or without poly(A) for cDNA synthesis.

Question 11

What are oligo dT primers?

Answer 11

String of 12-20 deoxythymidines that anneal to poly(A) tails of eukaryotic mRNAs.

Question 12

What is HPLC?

Answer 12

Purification over a column. Just need desalting and maybe this for standard PCR. In special cases, PAGE.

Question 13

What factors need to be considered when trouble shooting primers?

Answer 13

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.

Question 14

What to consider with nucleotides?

Answer 14

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.

Question 15

What amount of Mg++ is suggested?

Answer 15

0.5-3.5 micro molar. Too low Taq won’t work. Too high mispriming.

Question 16

What factors to consider with DNA polymerase?

Answer 16

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.

Question 17

What extras can be added to PCR?

Answer 17

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.

Question 18

What is quantitative PCR?

Answer 18

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.

Question 19

What are reverse transcriptases for?

Answer 19

Forming cDNA libraries. Contain DNA copies of mRNA from cells tissues.

Question 20

What are Taqman probes?

Answer 20

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.

Question 21

What is SYBR green I?

Answer 21

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.

Question 22

What are the advantages of TaqMan?

Answer 22

Increased specificity. Use when the most accurate quantitation of PCR product accumulation is desired. Option of detecting multiple genes in the same well.

Question 23

What are the disadvantages TaqMan?

Answer 23

Relative high cost of labeled probe.

Question 24

What are the advantages SYBR Green I?

Answer 24

Relative low cost of primers. No fluorescent labeled probes required.

Question 25

What are the disadvantages of SYBR green?

Answer 25

Less specific- only primers determine specificity. Specific and non-specific double stranded PCR products generate the same fluorescent signal upon binding SYBR Green I dye. Not possible to multiplex multiple gene targets.

Question 26

What is digital PCR?

Answer 26

Used for nucleic acid detection and critically quantification. Estimates absolute numbers of molecules through statical methods.

Question 27

How does digital PCR (dPCR) work?

Answer 27

By dividing a sample of DNA, cDNA or RNA into many individual microreactions (droplets). Some contain one or more molecules while others contain none. Each microreaction undergoes PCR amplification and analysis separately.

Question 28

What is dPCR ideal for?

Answer 28

After counting the positive microreactions, statistics can be used to then determine the absolute quantity of the target molecule rather than comparing to standard of known concentration. Ideal for rare allele detection.

Question 29

Look at the summary table.

Answer 29

Look at the summary table.

Question 30

What is RFLP restriction fragment length polymorphism?

Answer 30

Nucleotide sequence variations in a region of DNA that generates fragment length differences according to the presence or absence of restriction enzyme recognition sites.

Question 31

What is VNTR variable number tandem repeat?

Answer 31

Sequences that are repeated multiple times and the number of repeats varies from person to person.

Question 32

How is VNTR and STR (tandem repeats) used for finer print analysis?

Answer 32

These genes usually occur in introns which are not under evolutionary pressure, more variable in the population. Amplified with PCR and run on agarose.

Question 33

How is PCR used in genealogy?

Answer 33

The number of differences in your haplotype and of another persons shows how many generations you would have to go back to find a common ancestor.

Question 34

What is your haplotype?

Answer 34

Group of genetic variants that are inherited together from a single parent.

Question 35

Where does mtDNA come from?

Answer 35

Mitochondrial DNA is passed down unchanged from mother.