CHEM 352—Biochemistry II—sp—polymerase-chain-reaction

Bill Fold, a first-year graduate student, has been interested in amplifying the human SOD1 gene from human chromosomal DNA by using the polymerase chain reaction (PCR). However, Bill has never designed primers for use in the PCR before. Seeking help, he found assistance from a sixth-year graduate student, Sandy Beach. Using the human reference sequence for SOD1 (NCBI accession number NC_000021.9) from chromosome 21, Sandy designed the following two primers to amplify the SOD1 gene:

Here's what Bill discovered.

Yes, these primers will amplify the SOD1 gene from start to finish, the entire coding seuqence. The GeneBank record for NC_000021.9 gives all the sequence details. Scroll down until you find the CDS (coding sequence) link on the left, click and the page will highlight the CDS in the sequence at the bottom. The forward primer and reverse primers match the begining and end of the CDS.
However, the sequence Bill is working from is chromosomal DNA, thus, in humans, the CDS is divided by five introns. The CDS record should highlight this. Also notice that the product length (8809 base pairs) from primer-BLAST is much longer than you would expect for a contiguous gene of a 154 amino acid protein. Bill might try amplfication from a cDNA library if he wants only the coding sequence.
Running primer-BLAST (input of accession number and primer sequences only, database core_nt, ogranism Homo sapiens) will check those primers against all human reference sequences. There are couple alternate products, but they all look like other sequences of SOD1. Including a sizable number of which are only the CDS, from mRNA or SOD1 assemblies.
Sandy discovered that the two locations for these primers are very different in GC content. Thus, to hit a common ToC, she varied the lengths of the two primers. The forward primer is shorter than the reverse primer. There is a bit of self complimentarity in the reverse primer (see GGG and CCC).

You've been studying wild-type superoxide dismututase (SOD1) in vitro and would like to compare that activity to the common form of the protein found in familial amyotrophic lateral sclerosis (ALS). The most common variant is a change of glutamic acid to histidine in superoxide dismutase. You have a system for expressions of SOD1 in E. coli. The region of the SOD1 gene around the codon in question is shown below, with the glutamic acid codon highlighted. You are considering using the four primer method to make this mutation. Design both the forward and reverse mutagenic primers necessary for changing the codon for glutamic acid to a codon for histidine. Also, detail the reactions you would run in the thermocycler (you can safely assume that you have amplification primers along with all other reagents).

	T	C	A	G
T	TTT PHE TTC PHE TTA LEU TTG LEU	TCT SER TCC SER TCA SER TCG SER	TAT TYR TAC TYR TAA STOP TAG STOP	TGT CYS TGC CYS TGA STOP TGG TRP
C	CTT LEU CTC LEU CTA LEU CTG LEU	CCT PRO CCC PRO CCA PRO CCG PRO	CAT HIS CAC HIS CAA GLN CAG GLN	CGT ARG CGC ARG CGA ARG CGG ARG
A	ATT ILE ATC ILE ATA ILE ATG MET	ACT THR ACC THR ACA THR ACG THR	AAT ASN AAC ASN AAA LYS AAG LYS	AGT SER AGC SET AGA ARG AGG ARG
G	GTT VAL GTC VAL GTA VAL GTG VAL	GCT ALA GCC ALA GCA ALA GCG ALA	GAT ASP GAC ASP GAA GLU GAG GLU	GGT GLY GGC GLY GGA GLY GGG GLY

Alright, there are two codons for histidine, CAT and CAC. As a first rule, you'd like design primers that make the smallest changes possible. Using primers that have one mismatch should be more reliable than primers that have three or more mismatches (yes, you could use this method to introduce sequence into the middle of the gene). As a second consideration, we should account for the codon bias in E. coli. The genetic code, for many amino acids, is degenerate and multiple codons can specify the same amino acid during translation. However, each codon matches with an anticodon on the tRNA during protein synthesis, and not all tRNAs are expressed at the same level. This naturally allows for another level of gene expression control. In E. coli, the histidine codon distribution is CAT (0.57) and CAC (0.43). CAT is the favored choice.

When designing primers with internal mismatches, don't be too concerned with melting temperature calculations as the mismatch will destabilize the double helix. That is, make sure you design a primer with a reasonable melting temperature, but know that it will be just a bit less in practice. Consider using less stringent conditions during thermocycling. See the PCR laboratory for more considerations of primer design.

Below is a potential pair mutagenic primers with the codon in question highlighted.

Forward (+) mutagenic primer:

GAA AAA GCA GAT CAT TTG GGC AAA GG

Reverse (-) mutagenic primer:

CC TTT GCC CAA ATG ATC TGC TTT TTC

The first detail to note is that there are other primer designs that might be equally (or more) functional; your design does not have to match these exactly. The mutagenic primers are complimentary to one another. After you've completed the design of the forward primer, the reverse is the compliment (many companies offering oligonucleotide synthesis will offer an easy method for ordering of the compliment; you often only need the one sequence and the check of a box to make the compliment).

The four primer method requires three reactions. The first reaction is an amplification using the forward amplification primer and the reverse mutagenic primer. The template DNA is the wild–type sequence of your gene. The second reaction is the forward mutagenic primer and the reverse amplification primer amplifying from wild–type DNA. These two reactions are independent and both reactions can be thermal cycled at the same time. The third reaction uses products from the first and second reactions as template (remember that these two molecules are complimentary along the sequence of the mutagenic pimers) and the forward and reverse amplification primers. In the early cycles, the complete gene segment will be made as DNA polymerase extends the products of reactions 1 and 2. As the concentration of this species builds, the amplification primers will greatly increase the concentration of the complete gene segment. Since this was generated from the mutagenic primers, it is your gene with one or more altered bases (two in this case changing the glutamic acid codon to a histidine codon).