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PCR & DNA Sequencing Biology 224 Instructor: Tom Peavy October 25, 2010 <Figures from PCR by McPherson & Moller> PCR= Polymerase Chain Reaction • • • • • • “DNA photocopier” integral tool for molecular biologists work horse versatile (many applications) not difficult to perform technically fast PCR components Template DNA Primers dNTPs (water, buffer) Thermostable polymerase 1) Template DNA is denatured (Denaturation phase; 94C) 2) Primers allowed to anneal to template; Tm of primers is important (Annealing phase; variable temperature) 3) Increase temperature to optimum for thermostable polymerase (Elongation phase; 68-72C) 4) Repeat the whole cycle starting at step 1 Sources of Template DNA Genomic DNA RNA isolation and cDNA Plasmid, bacteriophage, cosmid and artifical chromosome DNA Pathological and forensic samples Archaeological samples Technical Difficulties Mispriming – primers anneal to alternate sites and not to “correct” or targeted site Needle-in-a-haystack (Template in limited amounts) Mismatches allowed internally if annealing temperature is low (below Tm) Misprimed PCR products will continue to be amplified (PCR primers are incorporated into the amplimer at the terminal end and will thus serve as a perfect match for future PCR cycles; large amounts of PCR product accumulate if in it occurs in the early cycles) Artifactual products on agarose gels can arise from Primer-Dimer formation Examples of inter- and intra-primer complementarity which would result in problems: Annealing Temperature and Primer Design Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a DNA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is: Tm = 4(G + C) + 2(A + T)oC In setting the annealing temperature of PCR reaction: • As a rule of thumb, use an annealing temperature (Ta) about 5oC below the lowest Tm of the pair of primers to be used if a good yield of product is desired • Alternatively, if an increased specificity is desired, one can either Perform touchdown PCR (high-low anneal temp) The Tm of the two primers should not be different because it may never give appreciable yields of product due to trade-offs (annealing temperature appropriate for one but not the other) Can result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand. Note: Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long. Primer Length The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner (to avoid large differences) Another prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. Lengths are generally 17-25mers (rationale: there is a ¼ chance of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4,294,967,296, or 4 billion): Primers can be designed with engineered sites at the 5’end (e.g. restriction enzyme sites, mutations) Mismatches can also be designed internally to facilitate in situ mutations (change coding sequence or create restriction sites) Note: only use the annealing portion to calculate Tm EcoRI Degenerate Primers For amplification of sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: Degenerate primers= a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences. Need to examine all the options for particular amino acids with Respect to their codon degeneracy For the opposite direction (5’ end race) need to reverse complement the sequence! 5’ complement reverse 3’ 5’ 3’ CGN CTG TGN CTT ACC CTG TTT CCN CTT GTG CCN A C A C C A 5’ 3’ NCC GTG TTC NCC TTT GTC CCA TTC NGT GTC NGC A C C A C A Design of degenerate primers based on amino acid sequencing: If you do not know where the peptide regions are located in the gene, then need to design PCR primers in both directions and try various combinations Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases Increased degeneracy means concentration of the individual primers decreases (of which there is only one exact match) thus, greater than 512-fold degeneracy should be avoided. 5’ GTG TTC NCC TTT GTC CCA TTC NGT A C C A C (24mer) degeneracy= (1/4)2 (1/2)5 = 1/512 3’ Can use deoxyinosine (dI) at degenerate positions rather than use mixed oligos: dI base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo General Rules for Primer Design - primers should be 17-25 bases in length; - base composition should be 50-60% (G+C); primers should end (3') in a G or C, or CG or GC (prevents "breathing" of ends and increases efficiency of priming) - Tms between 55-80oC are preferred; - runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided; - 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; - primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided. Nested PCR Design two outside primers for the first reaction, Then use a portion of the first reaction as template in a second reaction using Internal ‘nested’ primers Multiplex PCR - uses multiple PCR primer sets to amplify Two or more products within single reaction - used for genotyping applications where simultaneous analysis of multiple markers is advantageous (or statistically necessary) - Can amplify over short tandem repeats (STRs) Short Tandem Repeats (STRs) AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Real-time PCR quantitation DNA Sequencing Sanger Method: Generating Read 1. Start at primer (restriction site) 2. Grow DNA chain 3. Include ddNTPs 4. Stops reaction at all possible points 5. Separate products by length, using gel electrophoresis Dideoxynucleotide chain termination method of DNA sequencing • Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel. • Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser. • In addition, can sequence directly from PCR products • Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment . This is an example of a good chromatogram showing well-resolved peaks and no ambiguities. Generally the first several hundred bases of a chromatogram will look like this. Start of a chromatogram showing peaks corresponding to unincorporated dye-terminators (dye-blobs) superimposed over and partially obscuring the real peaks. Region of a chromatogram fairly far along the sequence where some bases in runs of 2 or more are no longer visible as single peaks This is a region of a chromatogram where the traces have become too ambiguous for accurate base calling. While some parts of this region of the chromatogram can be useful for linking to existing sequences following manual editing, it should not be considered accurate.