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You are studying mutations in a bacterial gene that codes for an enzyme whose amino acid sequence is known. In the wild type protein, proline is the 5th amino acid from the amino terminal end. In one of your mutants with nonfunctional enzyme, you find a serine at position number 5. You subject this mutant to further mutagenesis and recover 3 different strains. Strain A has a proline at position 5 and acts just like the wild-type strain. Strain B has tryptophan at position number 5 and also acts like wild type. Strain C has no detectable enzyme function at any temperature, and you can't recover any protein that resembles the enzyme. You mutagenize strain C and recover a strain ( C-1) that has enzyme function. The second mutation in C-1 that is responsible for the recovery of enzyme function does not map at the enzyme locus. a) What is the nucleotide sequence in both strands of the wild-type gene at this location? b) Why does strain B have a wild-type phenotype? Why does the original mutant with serine at position 5 lack function? c) What is the nature of the mutation in strain C?
a) You also wish to synthesize a degenerate oligonucleotide primer that could be used eventually to clone DNA that encodes your mutant protein. Design this degenerate oligo, keeping in mind the rules we talked about in class. More information about designing degenerate primers and primers in general are located on the following page.
Write out the sequence of this oligo here
b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?
RULES TO FOLLOW for creating primers (Modified from the Thermo Fisher web site):
Designing degenerate primers:
Write out your amino acid sequence, label amino and carboxy termini
For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.
Now you need think about 5â and 3â considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5â end of a gene corresponds to the amino terminus of a protein. Using 5â and 3â labels write out a single strand DNA sequence corresponding to the your amino seq of interest. You may choose to start with only possible codon for each amino acid, or you can do all possibilities using the notation I showed you in class to account for the wobble position of codons.
Now make your DNA double stranded by filling in the opposite DNA strand; label the 5â and 3â ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.
If you havenât already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.
Realize that when a researched synthesizes a degenerate oligo that the final synthesis contains oligos with every substitution possible. The amount of degeneracy is defined by the number of different primer combinations in the mix. You can determine the degree of degeneracy by multiplying the number of changes present at each position together. If a position has no degeneracy then you multiple by 1 for that position.
Keep in mind that the trade-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants. Anything over 1024 fold degeneracy is at the upper limit of potential usefulness.
Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. This information is taken from the Fisher Scientific website and are used when actually designing an experiment. Here are some tips to consider when designing primers.
In general, a length of 18 nucleotides is the minimum necessary for efficient primer binding.
Try to make the melting temperature (Tmm) of the primers between 65Â°C and 75Â°C, and within 5Â°C of each other.
If the Tmm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding. This is called a GC clamp.
Typically, 3 to 4 nucleotides are added 5â of the restriction enzyme site in the primer to allow for efficient cutting. Restriction enzymes are ENDO nucleases, so they cut better when their recognition site is not on the end of DNA.
Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.