6 - PCR Flashcards
PCR primers
Must be long enough to bind the specific target DNA, usually 18 ro 22 nt. the longer the primer, the less change of primer binding to a random seq.
Must also be complementary to the beginning and ending points of the target DNA.
The total number of the bps and their order can also affect PCR efficiency. The more CG, the higher the Tm. The two Tms shouldnt be more than 5C apart, as this could lead to the complications below for one of the primers. CG content should be 40%-60%.
The annealing temperature is usually set 5C lower than the loewst Tm (either forwards or reverse strand). Thus, if the annealing temp is too high, the annealing cannot happen. If the annealing temp is too low, the PCR primers can bind to complemenraty refions outside the target DNA, and amplify the wrong part. This is called MISPRIMING.
Primer-dimer
if the primers in PCR have complementary bases, they could bind to each other, making a primer-dimer. These are stable, and lead to amplification of this rather than the target dna.
primer hairpin
If two segments on one primer are complementary, a hairpin could form
alternative polymerases
Most used: Taq-polymerase.
Pfu is used when there is need for increased accuracy, as it has proofreading.
Tli also has 3’-5’ proofreading.
What can be done of the target sequence is long?
Normal PCR: <5kbp.
long range PCR: 20-30 kbp.
1) increase extension/elongation time
2) add a mixture of polymerases. In short seq, the low rate of mispairing by Taq is ok, but in longer it is a problem that there is no proofreading. Solution: add Pfu or Tli, which can proofread what the Taq does.
3) long target seq are often damaged by heat. Lower the denaturation time from 1 min to 2-10 sec to protect the purine bases. Also lower temp for elongation/extension
4) buffer condifdion can be modified to provide better ionic concentrations for the alternative polymerases
Preventing mispriming
raising the annealing temp usually solves the issue
however, sometimes the mispriming happens during the reaction set-up. HOT-START PCR is a variant of regular PCR that reduces nonspecific amplification at the lower temperature steps od the PCR cycle, especially during the original setup. the key idea is to prevent Taq polymerase activity at low temperatures. Several different approaches have been used to block Taq polymerase with a reversible modification. the physical approach simply keeps the reagents apart until activated by heats, but this is cumbersome when doing large numbers of PCR reactions at one time. Other approaches inhibit DNA polymerase by Ab or blocking proteins that are released at high temperatures. These attach to the active site of Taq polymerase. These are also destroyed after the first step of PCR as they are unstable at high temperatures.
Adding sequences to PCR amplifications
DNA pol only rec the last 6 nt on the 3’ strand of each primer, and does therefore not really use the 5’ nt of the primer to start the DNA synthesis. The 5’ end mainly ensures proper binding, but the very 5’ end of the primer doesnt have to anneal to the template. This allows the design of tailed pCR primes (have additional sequences on the 5’ end that become incorporated into the final PCR product).
The tail seq can have many different uses. Could contain RE rec seq, which can be used to prevent overhangs that wuld be useful for cloning of connecting to anther piece of DNA. The seq could also contain a seq complementary to a different piece of DNA. These ends could be rec by the enzymes essential for homologous recombination, which would allow the PCR amplicon to integrate into a genome or other cloning vector. The uses for this PCR modification are really only limited by the length of PCR prmer that can be synthetized, but still provides useful modification.
Degenerate PCR primers
Can be used if f.ex a gene seq for one organism is known, and the corresponding gene from another organism is unknown.
degenerate primer: primers designed to have more than one possible base at particular positions within the seq. After synthesis, there are different primers, some will have one of the possibel bases, and others will have the alternative bases.
If the amino acid seq of protein is known, one could use that to design a primer. The primers can be synthetizes with mixtures of different bases at the third position.
The goal is to make many primers, add them all, and hope that at least one will be complementary to the right part of the target seq.
Inverse PCR
method for using PCR to amplify unknown seq by circulating the template molecule
can be used if a seq of part of a long DNA molecule (say, a chromosome) is known, and the objective is to extend the analysis into the unknown regions.
Makes the DNA into a circle. A RE cuts up- and downstream of a known seq. This results in a seq:
UNKNOWN - KNOWN - UNKNOWN
The teo ends will have compatible sticky ends, and are ligated. Two primers for the known region is used, and the synthesis will be clockwise for one and counter-clockwise for the other.
Reverse transcriptase PCR (RT-PCR)
Variant of PCR that starts with mRNA to make copies. First the mRNA is converted into a cDNA using reverse transcriptase, and then PCR makes multiple copies of the cDNA
cDNA = complementary DNA; version of a gene that lacks the introns and is made by the corresponding mRNA using reverse transcriptase
Needs a primer, providing the starting point for reverse transcriptase. the resulting DNA:RNA is denatures during PCR. the ssDNA is then used as a template for synthesizing dsDNA, called cDNA.
Very useful for studying euk genes, as they usually have introns.
Specific RT-PCR
- design a primer with a seq complemenraty to the mRNA. This is used by reverse transcriptase and dna polymerase.
- used for short seq, as full-length cDNAs are hard to get, esp from mRNA that is present only in very low amounts or unusually long. reverse transcriptase usually fails to reach the end of a long RNA template due to hinderance by RNA secondary structure, and thus the 5’ end is often incomplete. Obtaining a full-length cDNA can sometimes take multiple primer sets
converting an entire mRNA sample into cDNA
happens during the reverse transcriptase reaction.
must have nonspecific primer.
two options for creating this:
1) a primer that has a stretch of Ts (usually 18-20) can be used. This oligo(dT) primer specifically recognizes the polyA tail found on mRNAs, so when reverse transcriptase starts synthetis of the DNA strand complementary to the mRNA template, the final cDNA sample represemts more 3’ end seq.
2) use a random hexamer (= a primer that is six nt in length that is synthetized so that each nt can be any four bases. These are a mixture of primers that have every potential six nt seq, and are therefore compementary to any random 6nt seq of RNA). These bind at random along every mRNA in the sample, and prime vonversion of the mRNA into cDNA at any location. The final cDNA sample has a random sampling of all different regions of each and every mRNA that was in the original sample.
PCR in genetic engineering
two broad categories:
1) rearranging large stretches of DNA
2) changing one or two bases of a DNA seq
Overlap primer: PCR primer that matches small regios of two different gene segments and is used in joining segments of DNA from different sources.
overlap PCR reactions use a primer for front end of one gene, rear end of second gene, and overlap primer.
molecular sewing: creation of a hybrid gene by joining segments from multiple sources using PCR. Can amplify first, then join, or add all three primers and just get the finished DNA.
directed mutagenesis (kanskje se litt mer på det, s 186-187)
deliberate alteration of the DNA seq of a gene by any of a variety of artificial techniques.
Can be achieved by introducing mutations in the PCR primer.
Engineering deletions and insertions by PCR
1) a specifically targeted cassette is constructed by PCR. This contains both a suitable marker gene and up- and downstream seq homologous to the chromosomal gene to be replaced.
2) the engineered cassette is transformed into the host cell and homologous crossing over occurs. Recombinants are selected by the antibiotic resistance carried on the cassette.
barcode: a unique seq added to a cassette to mark the gene with a tag for subsequent analysis
index: a unique nt seq added to a cassette to mark the gene with a tag for subsequent analysis.
insertions or deletions.
