Cold Case DNA Analysis – The Progresses and Perils
Jul 2015

Cold Case DNA Analysis – The Progresses and Perils

Suzanna Ryan,

Owner/Consultant at Ryan Forensic DNA Consulting.

Last year, I was selected as a member of the Review Board of the American Investigative Society of Cold Cases. AISOCC is a “non-profit, non-biased, professional organization that will review your cold case free of charge” ( I was pleased and excited to begin reviewing cold cases as a member of the AISOCC Review Board since cold cases have always held my interest and, as a forensic DNA expert, I am well aware of the many changes that regularly occur in the field of forensic DNA analysis.

These changes include a great number of advancements in techniques and sensitivities that have the ability to greatly impact cold cases in a positive way.

This article seeks to explain some of the improvements and advancements in forensic DNA analysis, but also to caution the reader regarding some of the pitfalls to be aware of when performing DNA analysis on cold case evidence samples.

The Progresses:

There may be evidence items that were collected and placed into evidence for one reason (examples include a latent print examined for fingerprint identification, a shoe examined for footwear impressions, a knife collected for wound comparisons, a gun examined by the firearms section, a screwdriver examined for tool- mark comparisons) that may have yielded little or no useful information during the original examination but may yield a DNA result. Each of the items listed above could very well contain the DNA of a person who touched or handled the item and then left it at the crime scene.

Investigators should be aware of the seemingly endless list of items that may be contain enough “handler” DNA to yield a DNA profile. Even though an item was not collected for a particular purpose, like “touch” DNA analysis, this does not mean that it cannot be useful today. The sensitivity of the amplification kits used in most forensic laboratories is truly amazing. The Identifiler Plus amplification kit and the PowerPlex 16 HS kit are designed to overcome inhibitors and work well with low-level samples. Consider that the typical target amount of DNA for an amplification is somewhere around 1 nanogram, or ~152 cells, and that these kits routinely yield results with much less sample than this, and you will have an idea of just how little DNA-containing material is needed for amplification.

New Methodologies:

The types of forensic DNA analysis in use today include techniques that allow one to examine only the male DNA in a sample,amplification kits that are designed to work with degraded, inhibited, or very low- level samples, techniques that can yield a DNA sequence from hair samples without a root, and even amplification kits that can identify animals.

  1. Male specific quantitation systems and Y-STR analysis can reveal whether any male DNA is present in a sample. If a very small amount of male DNA is present in the presence of a much larger amount of female DNA, typical results in the past would have yielded only the female DNA profile as the male DNA would be masked by the much larger amount of female DNA. However, Y-STR testing can detect male DNA even in the presence of 100’s or 1000’s of times more female DNA. Examples of cases that can benefit from the use of Y-STR analysis include rape cases involving perpetrators who are vasectomized or otherwise do not produce sperm cells, cases of rape without ejaculation, female victim’s fingernail scrapings, swabs from ropes or bindings used to tie up female victims, or any other case involving a female victim and one or more male suspects.
  2. Mitochondrial DNA analysis, while not a brand new technology, may not have been in use at the time of the initial crime. One of the most common sample types for mitochondrial DNA testing are hairs. Hairs located at a crime scene or on a victim’s or suspect’s clothing may have been subjected to hair comparison analysis in the past, but, unlike mitochondrial DNA typing, hair comparisons are not an exact science. The results of mtDNA testing determine whether an individual can be excluded as a contributor of the hair with 100% assurance. A matching mtDNA profile indicates that the hair could have come from the person being tested, or from a maternal relative of that individual. As such, mitochondrial DNA testing can be very useful in missing person’s cases, as well as criminal cases.
  3. MiniSTR testing is a technique that was developed for use with the bones collected from the World Trade Center after 9/11. Some of the bone samples from this site were badly damaged due to heat, mold growth, and other factors such that traditional STR typing, as sensitive as it is, just could not yield a DNA profile. MiniSTRs examine 8 areas on the DNA molecule that are the most likely to show difficulty in typing during traditional STR testing. The Minifiler STR kit currently on the market is extremely sensitive – yielding results with much less than half a nanogram of DNA in many cases – and is designed to work with difficult samples that are degraded or contain inhibitors to the PCR reaction.
  4. Animal hairs are often found at crime scenes, in vehicles, or on victim’s or suspect’s clothing. STR typing for dog and cat hair is now available at some (typically private) DNA laboratories. While hairs can be very transitory in nature, and easily transfer from one area to the next, the presence of a dog or cat hair that matches the suspect’s dog or cat on a victim’s body can be very useful information, especially if the two individuals did not know each other. Similarly, an animal hair matching the victim’s pet that is found in the trunk of the suspect’s car, for example, may help show that the suspect transported the victim in his vehicle (again, assuming that the suspect and victim do not know each other). This might not prove the case of and by itself, but it can let the investigator know if he is looking in the right direction.

Even if some of the evidence for your case has already been tested by a forensic DNA laboratory, it might still be worthwhile to pursue additional DNA testing. First, review what testing method was performed and whether the newer techniques discussed above could benefit your case. This is especially true if methods other than STR analysis were performed. Short Tandem Repeat analysis has been the standard in most forensic laboratories since around the late 1990’s or early 2000’s. So, if the evidence in your case was tested prior to this, there is a good chance that older testing methods, such as HLA-DQalpha, or even RFLP typing were performed. If this is the case, even if results were obtained, those results are not part of the National DNA Database. This means that if a foreign profile, assumed to be from the perpetrator of the crime, was developed using the older testing methods, this profile is not being searched against the 10.6 million convicted offender samples, 1.7 million arrestee samples, or 527,000 forensic unknown profiles currently housed within the database.

Second, even if it appears that no sample remains after being previously tested – it may be possible that some sample does remain. For example, if all of the victim’s vaginal swab has been consumed in prior testing, but the lab retained the original packaging, the sensitivity of today’s tests are so great that it might be possible to swab the interior of the swab package and get a DNA profile due to transfer of DNA from the swab to the interior of the packaging. Likewise, if a hair root was identified and tested for the presence of DNA with negative results, it is likely that a portion of the shaft still remains and could be tested with mitochondrial DNA.

The Possible Perils:

Now that many of the ways that DNA might benefit a cold case have been discussed, it is prudent to also discuss some potential problems that may arise when dealing with cold cases in general and cold case evidence in particular.

Risk of Contamination Increases
The possibility of contamination of cold case samples cannot be over-stated. The older the case, the less likely that care was taken by investigators, crime scene personnel, or even laboratory analysts to avoid contamination. This is not an indictment of the people involved in the case – it is simply an unfortunate side-effect of the fact that DNA testing has evolved so rapidly. Items that would never have even been considered for DNA testing 10 years ago are now routinely processed in the lab. Prior to the advent of the Polymerase Chain Reaction (PCR) a bloodstain the size of a quarter needed to be available for DNA testing. Now, a bloodstain the size of a pin-head (or even less) is needed. And, it is not just blood, semen, saliva, or other body fluids that are being examined in the lab. Labs are also testing skin cells.

Although it might be possible to get a DNA profile from evidence that has already been processed by another section of the laboratory, one must be aware that techniques to avoid contamination might not have been in place at the time the evidence was collected. For example, while we now know that DNA profiles can be obtained from fingerprints – if a print was processed using powders and brushes that had been used on other items of evidence first, it is quite likely that the brushes and powders are contaminated with DNA. If the firearms section test-fired the murder weapon to perform comparisons without wearing gloves, the DNA profile that will be developed from the surface of the gun is probably going to match the firearms examiner. If the medical examiner fails to properly clean the fingernail clippers between bodies, artificial mixtures of DNA can be created. Even if the item was only processed by the DNA section to begin with, there could be contamination issues. Prior to the advent of PCR, different protocols and different levels of cleanliness were in place because it just wasn’t possible to get a DNA profile from a small amount of DNA.

Above, we discussed the transfer of DNA from the swab itself to the packaging and mentioned that DNA material may remain on the interior of the swab packaging even if no swab material is left. That is a good thing, in terms of possibly having additional material to work with. However, this same transfer can occur if items are packaged together. For example, if all the clothing items from a victim are packaged in the same large brown paper bag, it is possible for DNA to transfer to different areas on the same item of clothing as well as to different items of clothing in the same bag. This, in turn, can lead to problems interpreting the results or even reconstructing the crime.

Another potential avenue of contamination is if the case has already been to trial. It is possible that evidence items were opened and handled by attorneys or jury members thus leading to another possible contamination scenario.

Dead or Missing Suspects
The older a cold case is, the more difficult it can be to track down witnesses and persons of interest. In fact, it could even be that a possible suspect has passed away since the crime occurred. In this case, if you obtain a DNA profile from your evidence, you may not have a reference standard to compare it to. Short of exhumation of the body, it may be impossible to compare a deceased suspect’s DNA profile to evidence associated with the crime. However, some other avenues may exist to obtain a reference DNA profile. One of the best methods is to check medical records for biopsy or other specimens. For females, Pap Smears may have been retained by the doctor’s office. Medical specimens make great secondary DNA standards.

If no medical specimens exist, a secondary standard may be available if the personal belongings of the deceased individual are still in existence. For example, toothbrushes, hairbrushes, glasses or contacts, pipes, canes, hats, etc. can all yield DNA profiles. Another possible specimen retained by family members are baby teeth. If none of these are available perhaps a letter confirmed to be from the individual can be located and tested for DNA (flap and stamp area).
It also may be possible to “recreate” the suspect’s DNA profile using family members. The more family members that are available the easier this becomes. At a minimum, the Y-STR or mitochondrial DNA profile from a relative could indicate if the person of interest can be eliminated from suspicion or not.

Storage Problems
Another cold case peril includes improper storage of evidence items. For example, cold case evidence may have been stored at room temperature, or even worse, in a hot storage shed somewhere. Even if the items had been refrigerated or frozen, it is possible that at some point over the years the electricity went out, or the fridges or freezers broke down, leading to moldy, degraded evidence. While modern forensic testing can overcome some degree of degradation, the evidence may be too far gone to obtain a profile.

This leads us to another possible issue. The older a sample is, the more likely it is to show signs of degradation (especially if it hasn’t been stored properly). In some cases, no results may be obtained. In other cases, a partial profile may be obtained. If enough loci are developed, the partial profile can still be searched in the State DNA database. If a partial profile is obtained and searched at the State level, one must be aware that it is possible (especially given the large number of DNA profiles that are now in the database) that a “coincidental” match could occur. DNA testing does not look at the entire DNA molecule, instead only 15 areas are examined. If a full profile is developed, the probability of the DNA coming from someone other than the person it matches is exceedingly rare, if not impossible. However, if a partial profile is obtained, and a cold hit occurs in the database, the investigator must be aware of the possibility of a coincidental match – especially if there is no other corroborating evidence. In this case, more investigation will be required to try to eliminate or include the suspect as the true perpetrator of the crime.

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