#ASKtheProf - Have any repair methods like ligase been applied to the burnt samples before amplification?

A simple ligase treatment can improve DNA recovery in highly degraded remains.

You #ASKtheProf, he listens! Last week while in Denver, USA at the #ISHI34 forensic conference, Dr Vanessa Lynch had the chance to ask Prof. Bruce Budowle himself to answer this week’s excellent question by Ryan Lehto, PhD which focuses on ligase treatment, highlighting the importance of DNA quality and quantity for effective analysis.

29 September 2023

By Prof. Bruce Budowle

What a great question Ryan Lehto, PhD ! Presumably, the reason you posed this question shows that you appreciate the fact that DNA analysis is only as good as the quality and quantity of DNA available. The concept being that damaged/fragmented DNA from challenged samples could be repaired so more template DNA may be accessible for downstream analyses.?

A DNA ligase is an enzyme that catalyzes the formation of a covalent phosphodiester bond joining two DNA strands. I am not aware of any forensic case or burned victim case where ligation was employed to overcome highly fragmented DNA. However, there is a commercial kit, “PreCR? Repair Mix” (New England Biolabs), which is an enzyme cocktail designed to repair a wide range of damages to DNA before being amplified by PCR.?

One of the enzymes in the cocktail is a ligase. This ligase is quite effective at repairing nicks in the DNA, but it does not ligate fragmented DNA (i.e., blunt ends cannot be joined). Thus, creating longer template fragments cannot be achieved. Nonetheless, nicks in DNA can inhibit the amplification of target DNA, so there may be some types of evidence in which DNA templates may be improved with nick repair.

Existing studies

There have been some studies assessing the use of PreCR on forensic samples. One such study was published by Robertson et al. (Assessing PreCRTM repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification, Forensic Science International: Genetics 12 (2014):168–180). In their study, they had some success improving the recovery of results from intentionally damaged DNA under controlled conditions.?

One of the challenges with repairing DNA is that in the real world DNA can be damaged in multiple ways, and such repair mechanisms may work for some samples (or parts of the DNA in a sample) and not for others. Currently, it is difficult with routine operations to predict how samples are damaged; thus, successful outcomes can be spurious. The authors found their results promising but recognized that more research is needed.?

Value of ligation

Ligation, however, can be used in other ways to improve the typing success of highly degraded/damaged DNA. As an example, I recommend reading the paper by Sundararaman et al. (A method to generate capture baits for targeted sequencing, Nucleic Acids Research 51 (2023):e69). Just as was suggested by Ryan Lehto, PhD who posed the ligase question, these authors sought to enrich target DNA. Prior to sequencing, they used hybridization capture of intended targets by synthesizing and employing large-scale baits. Baits are essentially oligonucleotide probes that hybridize to targeted regions to isolate the desired markers from among myriad other DNA molecules.?

For example, one could isolate human DNA housing identity testing single nucleotide polymorphisms (SNPs) from interfering microbial DNA in bone samples. Sundararaman et al. developed a methodology, “Circular Nucleic acid Enrichment Reagent” (CNER), to create such baits rapidly, in large abundance, and likely rather cost-effectively.? The CNER method circularizes target template fragments “via splint-ligation.” Circularized templates can then be amplified by rolling circle amplification (RCA), which creates long amplification products containing many tandem copies of the target region (i.e., the circular molecule). These products are digested with restriction enzymes into baits (or CNER probes) in microgram quantities. Thus, ligation plays an essential role in creating the circles to serve as templates for probe generation.? Capture probes enrich challenged samples so that subsequent massively parallel sequencing of targets of interest can be more successful.

So while ligation has not been used, to my knowledge, in routine casework, capture hybridization has been reported for the enrichment of DNA templates by, for example, the Armed Forces DNA Identification Laboratory for analysis of some of their most challenging human remains cases (read Gorden et al., Extended kinship analysis of historical remains using SNP capture, Forensic Science International: Genetics 57(2022):102636). We can anticipate that these cutting-edge methods will be developed further to improve successful analyses of the most challenging of DNA samples, and ligases will play a part.

Thanks again Prof. Bruce Budowle for taking the time to answer this question. If you or your team have any other questions to #ASKtheProf please send them to us by clicking on this link or posting them as a comment below this article.