What is degenerate PCR?
Where the Y=C or T, R=G or A, N=G, A, T or C.
The more wobbles you introduce in the PCR primer the more degenerate it gets.
(The degeneracy of the primer is produced during DNA synthesis, you do not need to order 256 different primers to get a 256 mix, that's a lot of paper work!! and expensive).
Degenerate nucleotide codes: R=AG, Y=CT, M=AC, K=GT, W=AT, S=CG, B=CGT, D=AGT, H=ACT, V=ACG, N=ACGT.
Why use degenerate PCR?
Degenerate PCR has proven to be a very powerful tool to find "new" genes or gene families. Most genes comes in families which share structural similarities. By aligning the protein sequences from a number of related proteins you can find which parts of the protein is conserved or which is variable. Based on this information you can find conserved protein motifs which can be used as a starting point to make degenerate PCR primers.Degenerate PCR can be used to "solve" a number of problems.You have isolated a protein and have managed to sequence some amino acids from it. You want to find the corresponding gene!! (Why not try with degenerate PCR!).
You have found a human gene and want to clone the homologue gene from i.e. Mouse or Drosophila. (Of course can you try with low stringency hybridizations, but how many false positives do you have to sequence before you find the correct one?).
You have found an interesting gene in yeast / C. elegans and want to find the human homologue (if it exist). (Why not try degenerate PCR!).
Phylogenetic and evolutionary studies of genes. I.e. you can find specific orthologous genes from a number of related species and compare them. (This type of information can reveal potential active sites, regulatory regions and much more).
In studies of gene families. (I.e. how many members of the Rab family exists in green algae, do they differ when compared with the higher plants?).
This is just a few examples of what kind of problems you can apply this technique for.Technical comments.
Requirements. (What kind of sequence information do you need to get started).
Two blocks of conserved amino acids / DNA seq. The length of the primers should be a minimum of 20 bp.
| The protein motif does not have to be 100% conserved. Sometimes a partially conserved protein motif is sufficient. Examples of common found substitutions are Glu <--> Asp and Arg <-->Lys. If you use the degenerate "codon" GAN, it covers both Glu and Asp. Similar if you use the MGN codon, (M=C or A), where you know there should be a basic amino acid (Arg or Lys), the MGN codon covers partially the Lys codon AAR. If there is a Lys residue you will however have a G/T mismatch in the number 2 base. This is normally no problem as long as this mismatch occurs in the middle or the 5' part of the primer. (Remember your biochemistry, the enol form of thymidine can base pare with guanine). |
The N-terminal part of a protein (obtained from protein sequencing) often gives enough sequence information to be used for degenerate PCR.
| If the the N-terminal sequence is 20-30 amino acids, it is often possible to make two degenerate primers, and you can PCR up a 50-90 bp cDNA fragment which you can use as probe to screen a cDNA library. Alternatively you can make two degenerate primers and try a 3' RACE, to amplify the rest of the cDNA. Hint: The easiest way is normally to PCR up a fragment of the N-terminal, sequence this fragment and then make specific primers for 3' RACE. |
cDNA or genomic DNA?
In general cDNA works best as template DNA. Lower complexity of DNA, (in eukaryotes a small percentage of the genomic DNA encodes proteins).
The size of the PCR fragment is "predictable" when you use cDNA, because there is no introns.
Genomic DNA can be used as starting material if you are uncertain that your gene is expressed in the tissue or development stage you have chosen. The drawback is that you often have to sift through a lot of junk DNA before you find your gene. Another potential problem with genomic DNA occcurs if your PCR primers are based on protein / cDNA alignments and one of your primers span a exon intron junction.
How degenerate can PCR primers be and still function?
How to choose the PCR conditions.
Try "standard conditions" with slightly lower annealing temperature, 35-50 cycles.
If negative by "standard conditions", run the first 4 PCR cycles at 5-10 C lower than "recommended", i.e. 42-46 C. (PCR primers with multiple mismatches will be extended, and hopefully some stick to your gene).
If the primers are very degenerate (512 mixes or more), competitive inhibition can give problems. (Primers bind the correct template but are not extended by the polymerase because of unstable 3' ends). This means that the first PCR cycles are very inefficient, and you some times have to run 50 cycles PCR just to see a faint band of your gene.
Remember not to use a DNA polymerase with 3-->5' exonuclease activity, (PCR primers will be degraded). Taq. polymerase works OK.
What types of genes is "easy" to find by degenerate PCR?
Implications:
The conserved amino acids you try to design the PCR primers after is composed of mainly Ser, Arg and Leu. (These are the amino acids which gives most wobbles). This can sometimes give primers which are so degenerate they amplify anything, normally just a lot of garbage.
The region you try to amplify is to big. As a rule of thumb, avoid PCR products larger than 1000 bp.
The organism you try to amplify the fragment from has a very high GC content. This often poses trouble and you end up amplifying a lot of incorrect fragments.
The organism you try to amplify the fragment from has a very low GC content and you have designed your primers too short, (high TA content gives a low melting point for your primers).
The gene you are looking for do not exist in the organism you have chosen. There are a few examples of "dinosaur genes" genes which have disappeared in certain lineages during evolution. (For instance Rac genes in S. cerevisiae).
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