“DNA is the fingerprint of the 21st century”. — John Walsh
DNA degradation is a challenge that forensic scientists have faced from the pioneer decades of DNA fingerprinting, to which the only responsible factors are time and environment.
Widespread damage to DNA is imminent post-death due to a variety of reasons which are hard to control if left alone. Studies show that in just three weeks, DNA in certain tissue samples when exposed to the environment (such as in water) become severely damaged and nearly unrecoverable. Many of these samples exhibit problems such as allele loss, oxidation, bacteria attacks, and cannot be amplified by PCR due to PCR inhibitors, which will all be discussed later on, but first it is essential to understand exactly how important DNA is for many fields, such as genomics, personalized medicine, forensics, etc.
The Role of DNA
DNA plays an important role in science due to one key fact: it is what makes us who we are. By analyzing DNA, we have the potential to look not only into the past using ancestry markers, but also into the future, such as when wanting to know which diseases you are more prone to.
Also of major importance is the fact that our DNA is like a fingerprint (fingerprints are actually formed from DNA), in the sense that our genetic code to only unique to us. This is a key factor in the field of forensic biology, which looks at how we can associate different people to different items, or locations for example, using our own biology as evidence.
All DNA in our cells are composed of four nucleotides, Adenine (A), Thymine (T), Cytosine (C ), and Guanine (G). These nucleotides pair with each other to make up the typical double helix structure we know of when we think DNA. A’s and T’s pair together, and C’s and G’s pair together, which means, if we know what one single stranded piece of DNA looks like, we can easily map out the other strand.
This is the basic logic scientists use for the sequencing of DNA, which is also the method used for differentiating your genome from mine.
What exactly are STRs?
STRs, or Short Tandem Repeats is a region of DNA composed of a short string of repeated nucleotides. For example, in a certain part of my DNA, I might have the pattern ACACACAC composed from our basic nucleotides. Since STRs vary from person to person, you, however, might have the pattern ACAC in the same section of DNA.
When sequenced and analyzed by forensic biologists, it is these key differences within our STRs that connect us with a piece of DNA from a crime scene, for example. Other markers scientists look for when analyzing DNA include the different variations of SNPs.
What are SNPs?
SNPs (pronouned “snips”), or Single Nucleotide Polymorphism are variations at a single base pair of a certain piece of DNA. Since as humans our DNA is 99.9% the same, these single variations are very important for the differentiation of one person to another.
SNPs are mistakes made by cells during synthesis (copying) of DNA. Sometimes, single nucleotides are taken out, added, or substituted with another base. When nucleotides are substituted, SNPs are created, and can then be sequenced and analyzed. In a typical human genome, there are around 10 million SNPs, which all vary from one person to the next.
How is degraded DNA Recovered?
PCR inhibitors (collogen, heme, calcium ions, etc) are substances responsible for affecting taq polymerase or binding DNA (or a combination of both) within a PCR amplification, which ultimately results in it’s failure. As time goes by when DNA is left in the environment, these inhibitors are more likely to be present within samples of DNA. Without PCR amplification, it is difficult to obtain a DNA fingerprint, or in other words, an STR of the DNA being studied (because STRs are used a lot more than SNPs for standerd analysis as of today).
Degraded DNA in itself is hard to recover due to the inhibition of PCR, which inhibits amplification of DNA. However, trace amounts of DNA may be recoverable within larger amounts of degradation, in which STR markers may be of use. Traditionally, forensic scientists translated STR markers into miniSTRs so that they can preform more enhanced PCR cycles. MiniSTRs are created by moving the forward and reverse primers closer to the STR repeat region.
As scientists have observed, miniSTRs preform quite well on degraded DNA. As society advances, however, next generation sequencing methods, such as MPS (Massive Parallel Sequencing) with the Illumina MiSeq and other emerging technologies make it easier to analyze large amounts of STR markers at once.
With advanced technology, it is possible in the near future that degraded DNA may be easier to sequence, as interpretation of STR and SNP loci become more accurate. Since SNPs rely on a smaller amount of DNA needed than STRs, theoretically the use of SNP loci should give scientists a more accurate picture than when using STRs on degraded DNA.
The Significance of SNPs
The traditional and extensively used STR markers are the primary way to profile DNA today. However, there are other and potentially faster methods for profiling degraded DNA; One of which includes the use of SNPs, as previously discussed.
Only a single nucleotide is needed to be analyzed with SNP markers compared to over a hundred nucleotides needed with STR. This would automatically make DNA degradation less of a problem, however, a lot more SNPs (around 40-60) need to be tested to produce an accurate sequence. In addition, mutations are much less common with SNPs than with STR strands, which makes profiling easier.
With an SNP profile, scientists gain a much more elaborate picture of a certain patronage and disease susceptibility. With this, treatments and drugs can be tailored to known links between certain SNPs and resulting diseases, such as cancers, as a method of personalized medicine. With degraded DNA, scientists can discover ethnic origin or ancestry markers using Y-SNPs, and mitochondrial DNA (mtDNA) using SNPs.
Known disadvantages of SNPs
The biggest disadvantage of SNPs is that since the amount of SNP loci that need to be examined compared to STR loci is a lot more, data interpretation ultimately becomes more difficult. Trying to profile with SNPs on often rather small amounts of DNA may fail to produce a result.
Should the profile fail, with limited amounts of starting DNA due to degradation, an opportunity to repeat testing on lost loci may not be present.
Because of this, the argument which brings up a point about SNPs being useful on a small amount of degraded DNA may not always be valid due to the complexity of examination and interpretation. In addition, the standard for SNP loci has not been widely adapted yet, which makes SNPs used even less mainstream.
The Overall Relation Between STRs and SNPs in DNA Degradation
Currently, the most mainstream method for analysis of DNA degradation is by using miniSTRs. However, should more advanced and powerful screening technology be used in the future, it is hard to say which method (if not another one, such as using mitochondrial DNA) will become more mainstream.
Needless to say, it is important to understand the different strengths and weaknesses of these methods in order to see room for advancements, especially when working with degraded DNA, which is a heavy factor in criminal convictions. Obtaining a quick and easy way to restore degraded DNA can open many doors for forensic biologists, and for multiple fields, such as genomics.