BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu

Introduction of restriction sites

• It is possible to introduce restriction site sequences into PCR products by having these sequences incorporated into the 5' end of the PCR primer(s).

• The short restriction site sequence on the 5' end of the PCR primer will not hybridize, but as long as the 3' hybridizing region is long enough (i.e. its Tm is high enough; ~20 mer) the primer will specifically bind to the appropriate site.
• The PCR product will thus have an additional DNA squence at the 5' end which will contain the endonuclease restriction site.
• A similar or different restriction site sequence can be added via the other PCR primer.
• If the other primer has a different restriction sequence then the PCR fragment can be inserted in a directional dependent manner in a host plasmid.

The potential problems with this method include:

• There is no easy way to prevent internal sites containing similar restriction sequences from being cut when the end of the PCR product are cut
• Restriction sequences are inverse repeat sequences, thus the potential exists for primer dimer association and resultant non-productive annealing

Generation of half sites

• This method is similar to the method of introducing restriction sites, described above.
• The primary difference is that instead of the primer containing the entire restriction site sequence (say the six nucleotides of a six cutter) it will contain only the last three (and the other PCR primer will contain the complementary sequence for the first three).

The advantages of this method are:

• Typically internal restriction sites cleave with much greater efficiency (i.e. some sites if located at the ends of linear DNA never cut well at all)
• There is no need to gel purify linker fragments after digestion
• The DNA can be methylated (the half sites will not be). After concatenation the linkers will be cut but internal restriction sites will not

• A disadvantage is that the same restriction site is incorporated into both ends so the PCR fragment cannot be ligated into a host vector in an orientation dependent manner.
• Also, in this method 3' A overhang cannot be tolerated.

Blunt end ligation

• As stated earlier some thermostable DNA polymerases add a single dA residue onto the 3' end of the PCR product.
• There are three choices to be made when attempting to subclone without the use of added restriction sites within the primers:
  • Use a DNA polymerase which leaves the 3' strand blunt (e.g. Vent) and do a blunt end ligation (i.e. host vector was opened up with blunt cutting restriction endonuclease)
  • "Fix" the 3' A overhang by chewing back with Pol I, dNTP's.
  • Use the 3' A overhang to anneal and ligate to a "T" vector - a vector which has a single dT overhang on its 3' ends.
• "T" vectors can be made by opening up a vector, via a blunt cutting restriction endonuclease and ligating in a specific linker.
• The linker contains a restriction sequence for a restriction endonuclease which recognizes an interrupted palindrome and cuts in the internal region with a single 3' overhang.
• The linker will contain two copies of the restriction recognition sequence, the first with a sequence in the interrupted palindrome which leaves a 3' T overhang and the second with a sequence in the interrupted palindrome which leaves another 3' T overhang
• This may sound confusing, so here's an example of how to make a T vector:

Ahd I restriction endonuclease:

             |  G A C N N N N N G T C   C T G N N N N N C A G           |  

Ahd I will cut this sequence to produce:

  G A C N N N                 N N G T C   C T G N N                 N N N C A G  

We could design an oligonucleotide with two Ahd I restriction sequences, with slightly different sequences in the interrupted region of the palindrome, to give:

             |                     |  G A C N N T N N G T C G A C N N A N N G T C  C T G N N A N N C A G C T G N N T N N C A G           |                     |  

If this this were inserted into a vector, and the vector then was cut with Ahd I, it would have the following sequence at the ends of the linearized vector:

  -G A C N N T                       N N G T C-  -C T G N N                       T N N C A G-  

In other words, a 3' 'T' overhang at both ends of the vector. A PCR product, with 3' A overhangs could thus be inserted into such a 'T' vector

Adding promoters, ribosome binding sites, start codons, and stop codons

• The ability to add unique sequences to the 5' ends of PCR primers allows for short control elements to be directly incorporated.
• These can include a start codon or stop codon (3 bases), a promoter (~30 nucleotide region) or a ribosome binding site (~8 bases).

PCR Mutagenesis

Gene fusion

• This method is useful for joining overlapping regions of a large gene, or for the construction of chimeric genes.

Creation of deletions within a gene

• A very similar methodology can be used within a single gene for the production of a mutant gene containing a specific deletion:

• If the gene is contained within circular DNA (i.e. a plasmid) deletions can be constructed in a single PCR reaction with a single set of primers (this type of methodology is also known as "inverse" PCR).

Generation of point mutation(s) - i.e. base substitution mutations

• The generation of base substitutions can proceed along a similar route as with the deletion mutations.
• However, in this case the primers are mutagenic - there will be a mismatch, or mismatches, between the primer and target sequences.
• The mutagenic oligo will have a lower than expected Tm due to this mismatch(es).

Introduction of base substitutions via asymmetric PCR:

Insertion mutagenesis

• Short insertions (~1-6 basepairs) can be incorporated directly into a PCR primer, either internally, or at the 5' end.
• If the template DNA is linear and the desired site of insertion is not at the end of the template, then the entire gene (plus insertion) can be produced using asymmetric PCR or overlapping PCR (i.e. shown above).

• Large insertions can be accomplished by using a template (the desired insertion) for PCR with the primers having 5' sequences which are complementary to the region of insertion in the desired gene:

"Random" mutagenesis with PCR

• The PCR protocol can be modified so as to introduce mutations at random positions in the target DNA.
• The principle behind the mutagenesis is misincorporation of bases at "random" positions.
• Misincorporation by Taq polymerase, for example, can be achieved by adding Mn²⁺ to the reaction buffer, and decreasing the concentration of one of the four dNTP's.
  • At the sites in the template where the reduced base should be incorporated, there will be an increased probability of misincorporation.
  • Thus, the choice of base with diminished concentration determines the sites in the template which will potentially be mutated.
  • The misincorporated base is more or less random.
• The ideal Mn²⁺ concentration to add varies between 0.1 to 0.5 mM and is determined empirically. The relative concentrations of bases is 1 mM for each base, except the reduced base, which is typically present at a 1:5 or 1:10 ratio (i.e. 0.2 to 0.1 mM).

1998 Dr. Michael Blaber