6: Polymerase Chain Reaction Flashcards
What are the characteristics of PCR primers?
Short pieces of ss DNA that are complementary to the sequences at the ends of the target DNA segment => provides specificity.
3’ hydroxyl gr. are essential for DNA pol to initiate DNA synthesis.
Made by chemical synthesis of DNA.
Often 18-22 nt long.
Longer primer more specific for binding the exact target seq.
GC content between 40-60%.
Forward and reverse primers should not have complementary regions => prevents annealing to each other (primer dimer)
Should also avoid complementary regions on the same primers (will give hairpins)
Provide a short summary of the PCR reaction (Fig. 6.01).
- Sample of DNA that has the target region is isolated. Heating => ss DNA
- PCR primers anneal to the beginning and ending of the target region.
- DNA pol makes copies of the target region.
What are the components of PCR?
- Target DNA
- Two PCR primers
- Heat resistance (thermostable) DNA pol. Often Taq pol
- Supply of nucleotides, deoxy-nucleoside triphosphates (dNTPs)
- Buffer with optimal ion concentration and pH value.
What is a thermocycler, and what processes occur?
Machine used to rapidly and accurately shift between several temperatures in a pre-set order (for PCR).
Temperature alternates between 4 and 95 degrees.
Typically 20-40 (30) cycles with 3 temperatures per cycle:
- Denaturation (95)
- Annealing (60), time depends on primer length
- Extension/elongating (72), optimal temp. for DNA pol. Time depends of the length of target DNA.
What is the name of the final PCR copies?
PCR amplicons / PCR products
Only includes the original target region as the DNA sequences are shortened per cycle where the synthesis started.
What is the (main) determinant of a primer’s melting temperature?
Sequence
Different because A/T base pairs are held together by two hydrogen bonds, whereas G/C are held together with three.
More G/C => higher T_m.
Concentration of salts in environment, concentration of primers and target DNA, interactions between nt within primers themselves.
Melting temperature:
Temperature at which half of the primers are hydrogen bonded to the complementary seq in the target DNA and the other half is unattached.
How is the annealing temperature calculated, and what is the consequences of wrong temperatures?
Annealing temp should be set 5 degrees below the lowest T_m for forward or reverse primer.
Both primers should have fairly equal T_m (range of 5 degrees) to avoid that only one binds to DNA (no amplification)
Too high:
Primers will not anneal to target DNA => no amplification.
Too low:
Primers may bind to similar seq outside the target DNA => amplification from wrong region, mispriming.
Do you know any alternative polymerases to Taq polymerase?
Taq from Thermus Aquaticus
Pfu and Tli polymerases:
- Advantageous because it has proofreading 3’ to 5’ proofreading exonuclease activity
=> important when long range PCR is produced
What is long range PCR, and what modifications has to be made to amplify long sequences?
Long range PCR:
- PCR rx. used specifically to amplify longer target sequences than std. PCR.
- 20-30 kb
Modifications:
- Elongation/extension time is increased to give pol more time to make DNA.
- Use mixture of Taq and another proofreading pol.
- Denaturation time decreased from 1 min to 2-10 sec to ensure that the longer seq doesn’t denature.
- Temperature for elongation is lower.
- Buffer conditions modifies to ensure better conditions for the alternative polymerases.
How can mispriming be prevented?
Mispriming: annealing of PCR primers to seq outside of target DNA if temperature is too low.
Hot start PCR:
DNA structure that forms when two regions of complementary seq near each other anneal.
Reduces nonspecific amplification at the lower temperature steps of the PCR cycle by preventing Taq pol activity at low temperatures.
Can also use Ab or blocking proteins to inhibit DNA pol from binding to DNA. Unstable at higher temperatures.
What are “tailed PCR primers”?
PCR primers that have additional seq at 5’ end that become incorporated into the final PCR product.
Use of the “tails”:
- Contain restriction enz recognition seq => creates overhangs for cloning or connection to other DNA fragments.
- Seq complementary to other fragment => homologous recombination
…
What is a degenerate primer?
Primer designed to have more than one possible base at particular positions within the sequence. After synthesis, there are different primers, some will have one of the possible bases, and other primers will have the alternate bases.
What is inverse PCR?
Methods for using PCR to amplify unknown sequences by circulating the template molecule.
This will amplify the neighboring regions of the unknown seq.
Two primers in opposite directions.
What is reverse transcriptase PCR, RT-PCR?
Reverse transcriptase is an enz in retroviruses that converts (m)RNA to complementary DNA (cDNA).
Need primer.
DNA:RNA denatured during PCR stage -> ssDNA -> ds cDNA (amplified)
cDNA allows researching of gene expression under different environmental conditions without introns being present.
If an entire mRNA sample is to be converted to cDNA, the primers has to be nonspecific.
- Oligo(dT) primer with long stretches of T’s complementary to polyA tail of mRNAs.
- Random hexamer (6 nt) with 4^6 unique primers within the set as each nt can be found at each position. Prime conversion of mRNA at any location.
What is directed mutagenesis?
Deliberate alterations of the DNA seq of a gene by any of a variety of artificial techniques.
Can synthesize a PCR primer that carries the required alterations. As long as the primer is long enough to bind to the correct location on either side of the mutation, the DNA product will incorporate the change in the primer.
Target gene on circular plasmid vector. Mutational primer can be used with a primer that anneals to the opposite strand, upstream of the mutation.
To get rid of unmutated DNA:
Add methylation-sensitive restriction enz. Only unmutated contains methylated regions.
