PCR (Polymerase Chain Reaction), a relatively new DNA cloning technique, allows forensic chemists to work with DNA fragments that may be hundreds of years old. DNA can be notoriously difficult to work with, and decomposes quickly.
Using allied techniques in archeological analysis with PCR (Polymerase Chain Reaction), scientists pieced together the identity of an unknown skeleton from the 15th century: King Richard III.
A Brief Account of King Richard’s Last Moments
King Richard III met his end at Bosworth Field in Great Britain more than 500 years ago. His dead body, unceremoniously draped on a packhorse from the battlefield, was brought to Leicester and buried in the Catholic Church of the Grey Friars.
The church, as fate would have it, would later be razed, and the burial site became the fodder of speculation. Although the precise location of the burial site seemed lost, the memories of King Richard III’s reign survived the centuries.
When researchers unearthed the King’s burial site, it resembled a crime scene—a disturbed grave site that forensic investigators needed to investigate. The remains of the King revealed no coffin.
Finding the Right Amount of DNA for Analysis
When researchers found the skeletal remains, the scientists noted that it seemed to be in good enough shape to extract enough DNA for further lab work. Although the skeleton was more than 500 years old, researchers were able to extract mitochondrial DNA from the femur and teeth. (Mitochondrial DNA is the most abundant DNA in the human body.)
The DNA molecule is susceptible to various types of degradation processes that would have rendered the skeletal remains insufficient for DNA analysis if the remains were discovered 50 years later. Although the skeleton was intact (with the exception of missing feet) it was a mere 680 mm under the surface—or approximately 3 inches from the surface pavement. When the archeologists found the remains, they were dumbfounded by their luck; it was the first set of remains found in the area formerly known as the Grey Friars Church.
Correct identification of the skeletal remains as King Richard required that he had a present day ancestor to compare with his DNA. Although the King had no direct lines of descent, there were other lines of ancestry with which to compare his DNA—his cousins had ancestors survived to the present day. The DNA of his ancestors would serve as a template with which to examine the Richards’ cloned DNA fragments. The cloned or amplified fragments would result from the revolutionary PCR technology.
PCR: P(olymerase) C(hain) R(eaction)
The technique of PCR, first proposed in 1970 by researchers at the University of Wisconsin, builds complete segments of DNA from fragmentary building blocks. The fragments consisted of enzymes, fragmentary DNA, and small amounts of intact DNA combine to make (or synthesize) copious amounts of the desired DNA.[ DNA—deoxyribonucleic acid is the stuff of our genes. It is made of four fundamental building blocks (four different molecules). The four fundamental building blocks of life, combined with biochemical logic tell the body how to function: from our eye color to our skeletal frame. ]
The method of the researchers at the University of Wisconsin laid dormant for approximately 15 years. Then in 1983 a biochemist, Kary Mullis, pondered how to decrease the time spent synthesizing DNA. He knew that most chemical reactions do not go to completion because of temperature (energetics) and time constraints. Enzymes possessed the necessary (catalytic) properties to beat the limitations, and Mullis could produce results (that once took weeks or months) in hours. He and his co-workers published a landmark paper detailing how they were able to diagnose Sickle Cell anemia in fetus DNA and incorporate the technique into the “amniocentesis panel.”
In short, here is the technique:
(A) Source DNA is heated in a watery solution containing all of the ‘ingredients’ necessary for cloning the small target amount.
DNA, once heated in a watery solution, will lose its characteristic helical structure.
(B) The un-helicized source DNA combined with the building enzyme (polymerase) and fragmentary strand of target DNA will produce a completed longer strand (original target DNA plus source DNA) as the solution cools.
(C) The product is re-heated in the watery solution so it can un-helicize and the process re-performed.
An important key to the process is that while product is made—it is doubles in proportion during each cycle. The first double helix produces two double helix structures. The process synthesizes DNA exponentially (not linearly).
The Results of DNA Matching
Utilizing PCR allowed scientists examining King Richard’s skeleton the means to take a fragmentary amount of DNA and compare it to his present-day descendants. The group of researchers performing the analysis determined that the skeleton was the ‘right match.’
Interestingly, if one reads the journal account, the match was not greater than 99 percent. To those of us who might proclaim that the work is less than perfect—the workers took the most rigorous path of determination, and threw away data that may be skewed in favor of the identification of the remains as Richard III.
By taking a rough-hewn path, these scientists allowed historical, archeological, genetic, and biochemical analysis to meld into an organic interpretation of the last moments of King Richard III.
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