PreCR Repair Mix from New England Biolabs

In DNA, the most labile bond is the N-glycosil bond which binds the base (A, T, C or G) to the deoxyribose backbone. Hydrolysis of this bond results in the loss of the base leaving an apurinic/apyrimidinic (AP) site that eventually decomposes to a nick. When DNA is amplified using Taq polymerase and it encounters an AP site, that individual PCR reaction will stop. If there are enough AP sites, the PCR reaction will either fail completely or fail to amplify the entire product. Since most DNA is stored in either an H₂O based buffer or purified an H₂O, it can be reasonably expected that AP sites will be found in all stored samples and the older the stored sample, the worse the damage will be. With the integrity of stored DNA samples in the forensic world, it is very important to find ways to either prevent the damage from occurring or to restore the integrity of the DNA. There are many ways that forensic samples can be degraded to the point of being unusable, including environmental damage, excess heat, UV light exposure, etc. However, it is of utmost importance to be absolutely sure that DNA extracts treated for damage return 100% accurate STR (Short Tandem Repeat)* profiles.

The PreCR Repair Mix contains a cocktail of enzymes that are designed to “pre-treat” DNA extracts. This mix can repair many different types of damage, such as damage from excess heat, UV irradiation, high acidity, exposure to gamma irradiation and even damage caused by the extraction process itself. The damage that can be repaired by the PreCR Repair Mix are abasic sites, nicks, thymine dimmers, blocked 3’ ends, oxidized guanine and deaminated cytosine, but it cannot repair DNA fragmentation nor protein-DNA crosslinks.

The kit comes with UV damaged DNA to act as a control. Unfortunately, the kit only has UV damaged DNA and not DNA with nick or AP sites. Following the kit instructions, I have found incubating the DNA at 37⁰C for 30 minutes rather than 20 minutes and leaving the extract/PreCR Repair Mix overnight at 4⁰C to work better in my hands.

Repairs do not appear to affect STR profiles in experiments I have been performing. PreCR Repair Mix adds a random base at an abasic site which enables the Taq polymerase to complete its reaction and because it adds one base to a site on the template DNA that is missing one base it does not affect PCR product size and so the STR profile remains the same. DNA sequencing, however, cannot be used as a random base has been placed into the abasic site changing the DNA sequence of the product. So PreCR Repair Mix may change the sequence of a strand of DNA but will not change the length of it.

Correction from New England Biolabs:
PreCR does not insert a random base at an abasic site, it actually corrects the mistake. As the sequence is not altered, the repaired DNA can be used for sequencing.

So far, the PreCR Repair Mix appears to work well and DNA that fails to produce a good STR profile produce better STR profiles after treatment. These have all been performed using staff samples degraded in various ways. Significant validation needs to be performed on damaged DNA prior to it being treated for use in a forensic lab to be completely sure that no errors are made by the enzymes during the reaction. However, this kit so far shows significant promise.

*STR PCR is a method used to identify organisms and their susceptibility to antibiotics, as well as to perform kinship analysis of many of this planet’s animals by performing a PCR reaction and determining the number of repeats detected for a specific locus. Here it doesn’t make a difference if the repeat is CCCAGGT or if a PreCR reaction has changed it to CCGAGGT at one locus. What matters is that if the reaction changes the number of bases in a repeat as it will produce an unusual result especially if starting with a very small amount of DNA leading to the possible belief that this profile may have another source. One way to detect this is if many of your DNA peaks are ordinary loci and you have a third peak that is identified as being defined a variant that is one base longer or shorter than normal. Because this appears relatively rarely and I have yet to encounter it in my experiments, it does not seem to be common so it “should” only be seen in one locus out of several. In this case, the DNA should be PreCR’d again and see if the variant occurs again along with an untreated sample if that loci is not affected by damage.