The molecular characterization of microorganisms is achieved by microbial culturing techniques which yield enough nucleic acids for amplification and genetic analysis.
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Nucleic Acid Analysis
The primary bottleneck in this procedure is the inability to culture forensically relevant pathogens under laboratory conditions; moreover, nucleic acids may be low quality or damaged, rendering them incapable of analysis.
Despite this, several molecular biology tools employ high throughput methods, namely next-generation sequencing or microarrays, which make biological material amenable to genetic characterization. Such methods require the enrichment of the DNA target to attribute genetic components to an organism (genetic attribution).
Researchers Rachel Keiser and Bruce Budowle, of the University of North Texas, review capture and whole genome amplification strategies as methods of determining low-grade and low quantity-microbial nucleic acid sample analysis as published in Microbial Forensics.
The most traditional method of increasing the concentration of nucleic acid (nucleic acid amplification), is the polymerase chain reaction (PCR). DNA from the sample is fragmented and mixed with primers that bind to complementary bases that flank the fragments of interest.
In the case of low-quality and low-quantity DNA the primer is incapable of annealing, and as such, cannot extend the rest of the fragment region (called the amplicon). To overcome this limitation, a procedure called whole genome amplification (WGA) is employed to increase the available template. WGA is a method of multiple displacement amplification (MDA) that offers whole-genome replication and minimizes ‘amplification bias’.
This refers to the exponential amplification of certain amplicons over others due to the uneven mixing of oligonucleotide primers. WGA amplifies the entire length of the genome with high fidelity, minimal damage, and without biasing genomic regions.
The high fidelity is achieved by the employ of a f29 DNA polymerase, the enzyme responsible for copying the nucleic acid sequence, which has a processivity (how many bases of DNA copied before dissociation) of approximately 70,000 bases, as well as an error rate of 1 in 106 base pairs.
Moreover, f29 DNA polymerase possesses 3’-5’ exonuclease activity, which enables the accuracy of the copied sequence to remain high. MDA, however, is limited in its application, performing best with fragments of DNA >2000 bases in length with a large number of targets; forensic DNA samples which, have been degraded, however, do not fulfill these requirements.
Advantages in PCR methods
Alternatively, PCR-based WGA methods offer the benefit of depending less on the quality and quantity of DNA. Degenerate-oligonucleotide-primed PCR (DOP-PCR), is an example of a WGA PCR-based method that can analyze low-grade biological samples.
DOP-PCR can cover the genome with a single reaction; it is distinguished from PCR in that only one primer is used (contrasting with the two used in PCR), and this primer is comprised of highly defined sequences at both ends, which are separated by a random (degenerate) sequence between them. The primer is first used in a low-stringency cycle, in which the 3’ end of the sequence binds, or anneals, to several complementary sites on the target.
Once bound, the degenerate sequence then binds to start the process of DOP-PCR for WGA. This allows the binding of the 5’ end, which permits the high temperatures to be applied for the high-stringency cycle that follows.
Limitations of the PCR method
Despite some success in improving the typing (sequencing), of DNA samples at low quality, MDA is thought to be more robust. it offers processivity and higher fidelity, relative to DOP-PCR. MDA involves the annealing of two random hexamer primers to a template, which is then amplified using f29 DNA polymerase.
MDA requires intact single-stranded DNA, and so is not applicable for samples at low quality and quantity.
Outside of single-stranded DNA, a method called rolling circle amplification (RCA), was designed for the amplification of circular templates - such as those found in bacteria and viruses.
These generate linear tandem copies, which are linear and continuous copies of the circular DNA. However, if the circular molecule is compromised, then RCA is no longer a viable method suitable for the amplification of plasmid and viral DNA.
Circularization of single DNA can also be achieved. There are three methods for this process; of them, T4 DNA ligase and CircLigase II, are enzymic.
The third is molecular inversion probes (MIPs), an enrichment method that allows for specified circle-based target copying. CircLigase II is preferable as it does not produce concatemers - which is a DNA molecule made up of multiple copies of this same genome in a series. When's circularised, The DNA can be subject to RCA - this is because RCA is the equivalent of a continued extension of several oligonucleotide primers along with a circular template.
If the circular samples are shorter, the effect of lesions is less pronounced. In theory, all single-stranded DNA can be circularized and sequenced using shotgun sequencing; however, some targets may be at low concentration and samples need to be sequenced one at a time.
As it is not necessary to examine the entire genome to determine the identity of the microbe, it’s preferable to target specific sites i.e. those specific to a strain or species. To target them, a method of selection is needed.
Genomic partitioning also known as capture is one such method. This allows for the selection of specific gene targets from a pool of the genome itself as well as other microbial genomes. The capture method has been used successfully for ancient DNA and mitochondrial DNA targeting.
Both follow the same protocols; a reference library based on the targeted genomic regions is produced. This library is then fragmented and used to produce a set of RNA probes that will bind to the specific targets.
Any unbound DNA is washed away and only hybridized or targeted DNA remains. The final capture of the hybridized probe-DNA complex enables the sequencing of the target molecules only.
An additional circle-based approach uses padlock or MIPs. A MIP is a single-stranded oligonucleotide that contains two target complementary regions that are either side of a single nucleotide polymorphism (SNP).
These are regions of variation in an organism’s genome which serve as unique identifiers – and these serve as the selected marker. When the two target regions hybridize to the regions either side of the SNP, the probe inverts.
Following this, the gap between the two regions is filled with a polymerase and then ligated. The end-product is a circularized MIP in which the SNP is integrated. Following this, the circularized MIP dissociates from the probe, and exonucleases are used to digest any non-hybridized probes.
The MIP is then subject to cleavage at the probe release cleavage site, followed by PCR amplification - usually, the PCR primers sites are engineered into the MIP.
MIPs are specific as the probe ends lie close to one another to facilitate the process of circularization and ligation. They also ligate directly to the genetic material which is ideal when the DNA is fragmented and/or damaged.
MIPs are also amenable to multiplexing which allows simultaneous targeting of several SNPs in one reaction. One limitation associated with MIPs is the short fragment template requirement that accompanies the flanking regions. However, RCA can be used to amplify in cases where the circularized MIP does not yield enough DNA for sequencing.
Methods for MIPS are complex because contaminants are also amplified in the process. However, MPS is high throughput allowing DNA sequencing (although not at 100% efficient). Using a high throughput shotgun approach the intended targets can be discerned from another contaminant DNA.
When combined with bioinformatics tools, targets can also be identified at high coverage and speed. Taken together with its ability to perform simultaneous reactions in parallel, multiplexing is easy. Bearing in mind the lack of 100% efficiency, high throughput offers the detection of products, as well as quantifying the level of inefficiency present in the process.
This is because the off-target sequences will not align with the reference sequence used during bioinformatic analysis - although they will minimize coverage of the intended markers. MTS offers improved sensitivity of detection, throughput, greater discriminatory ability and better typing of degraded DNA.
The authors conclude that the molecular biology tools available currently have the power to genetically characterize low quality and quantity nucleic acids as found in microbial evidence from crime scenes.
Of those, enzymic circularization followed by capture and use of targeted probes for PCR, and finally MPS, are technologies capable of analyzing otherwise non-viable and difficult to cultivate microbial samples. By employing these techniques, the tools available to microbial forensic teams are widened.
Kieser, E. and Budowle, B. (2020) Chapter 13 - Select methods for the microbial forensic nucleic acid analysis of trace and uncultivable specimens. Microbial Forensics (Third Edition) (pp.195-205). Cambridge, MA: Academic Press