Restriction Fragment Length Polymorphism (RFLP)
This is the first DNA fingerprinting technique being utilized as a forensic tool. Portions of DNA molecule contain repeated sequences and they are referred as tandem repeats. It is accurately defined as a region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion. The significance of these tandem repeats is not known yet, but they are means of individualization which will be helpful in forensic investigation. A sample of DNA is being isolated and is being fragmented by restriction enzymes which will recognize and literally “cut” the DNA into many fragments when there is a specific short sequence occurs. Such process is referred as restriction digest. The resulting DNA fragments are then sorted out through agarose gel electrophoresis. Smaller DNA fragments will move at a faster rate along the plate than their larger companions. Then they are being transferred to a membrane using Southern blotting, named after its developer, Edward Southern. In order to visualize the result, a nylon sheet is treated with radioactively labeled DNA probe containing fragments complementary to the sample DNA fragments in a process called as hybridization. Next, the nylon sheet is placed against an X-ray film and are left exposed for several days. In the mean time, radioactive decay products will strike the X-ray film which will lead to the bands being observed after the film is taken to be processed. A diagram depicting the whole process is shown in Figure 2. It is interesting to note that each individual person has their own unique length differences of either a DNA strand or RFLP which will lead to the difference of bands distance being observed when a scientist is to compare the results of more than one DNA or RFLP samples.
RFLP DNA typing is the first scientifically accepted protocol in United States as a usage for forensic investigation involving DNA. Unfortunately, the utility was short lived as it was overtaken by a more revolutionary and efficient Polymerase Chain Reaction (PCR) technique during the mid-1990s. During RFLP short history, RFLP did the most startling impact related to the impeachment trial of US President Bill Clinton or better known as the Lewinsky scandal, in which President Bill Clinton was involved in a scandal emerged from having an affair with a White House intern, Monica Lewinsky. Ms. Lewinsky claims to possess a dress stained with president’s semen and the FBI Laboratory report showing seven-probe RFLP match obtained between president’s DNA and the stain, which basically represents a definite match between the semen’s DNA with president’s DNA as the combined frequency of occurrence of the seven DNA types found was nearly one in eight trillion.
This is the first DNA fingerprinting technique being utilized as a forensic tool. Portions of DNA molecule contain repeated sequences and they are referred as tandem repeats. It is accurately defined as a region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion. The significance of these tandem repeats is not known yet, but they are means of individualization which will be helpful in forensic investigation. A sample of DNA is being isolated and is being fragmented by restriction enzymes which will recognize and literally “cut” the DNA into many fragments when there is a specific short sequence occurs. Such process is referred as restriction digest. The resulting DNA fragments are then sorted out through agarose gel electrophoresis. Smaller DNA fragments will move at a faster rate along the plate than their larger companions. Then they are being transferred to a membrane using Southern blotting, named after its developer, Edward Southern. In order to visualize the result, a nylon sheet is treated with radioactively labeled DNA probe containing fragments complementary to the sample DNA fragments in a process called as hybridization. Next, the nylon sheet is placed against an X-ray film and are left exposed for several days. In the mean time, radioactive decay products will strike the X-ray film which will lead to the bands being observed after the film is taken to be processed. A diagram depicting the whole process is shown in Figure 2. It is interesting to note that each individual person has their own unique length differences of either a DNA strand or RFLP which will lead to the difference of bands distance being observed when a scientist is to compare the results of more than one DNA or RFLP samples.
RFLP DNA typing is the first scientifically accepted protocol in United States as a usage for forensic investigation involving DNA. Unfortunately, the utility was short lived as it was overtaken by a more revolutionary and efficient Polymerase Chain Reaction (PCR) technique during the mid-1990s. During RFLP short history, RFLP did the most startling impact related to the impeachment trial of US President Bill Clinton or better known as the Lewinsky scandal, in which President Bill Clinton was involved in a scandal emerged from having an affair with a White House intern, Monica Lewinsky. Ms. Lewinsky claims to possess a dress stained with president’s semen and the FBI Laboratory report showing seven-probe RFLP match obtained between president’s DNA and the stain, which basically represents a definite match between the semen’s DNA with president’s DNA as the combined frequency of occurrence of the seven DNA types found was nearly one in eight trillion.
Image from: Saferstein R. Criminalistics: An Introduction to Forensic Science. 8th ed. New Jersey: Pearson Prentice Hall; 2004.Polymerase Chain Reaction (PCR)
PCR stands a better laboratory technique compared to DNA RFLP. Its advantages are listed below:
Ability to amplify minute quantities of DNA, thus it can be used in identification of even saliva residues found on cigarette butts etc. in which RFLP technique fail to do so
RFLP required a longer DNA strand (thousand bases) whilst PCR only require shorter strands (hundred bases). Longer strands mean its higher vulnerability to be subjected for degradation and less stable vice versa.
PCR is an in vitro technique for amplification of DNA and it involves initialization, denaturation, annealing, elongation, final elongation and final hold steps.
Short Tandem Repeats (STR)
This is the latest method of DNA typing and is the most successful DNA profiling procedure. STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule. It consists of a repeating sequences ranging from three to seven bases and its entire strand is rather short, consisting of less than 400 bases in length which also means it is a stable and reliable sample. Let’s use TH01 as a DNA segment which contains repeating sequence A-A-T-G to explain the usage of this technique. TH01 is extracted and using PCR technique, it is being amplified and is then separated on an electrophoretic gel. The number of A-A-T-G repeats, which is a characteristic repeating sequence found in this STR, is determined by examining how far the it has migrated on the electrophoretic plate. Each individual has two STR types for TH01 in which one inherited from each parent of that individual. If one is able to make to copy an STR, one can conclude that there is definitely a trail left behind by that particular person in the crime scene. Also, one can detect the gender of the DNA contributor which interest the crime laboratories. The target here will be the amelogenin gene located on the two sex chromosomes, namely X and Y chromosomes. When amelogenin gene is amplified and separated by electrophoresis, two bands will be shown whilst females who have two X chromosomes will show only one band. Also, there is another STR marker, namely Y-STR which derives from Y chromosome only, makes detectives able to conclude the involvement of multiple males involved in a sexual assault when there is blood, saliva or vaginal swab originating from more than one male.
Mitochondrial DNA (mtDNA)
MtDNA is a more sensitive ways than nuclear DNA profiling in the sense that mtDNA is found to be abundant as there are more mitochondria than getting a nuclear DNA from a single nucleus in a cell per se. Unfortunately, all individuals with the same maternal lineage will have difficulty to be distinguished using this technique. Though more sensitive, mtDNA analysis is costly and time-consuming.
In mtDNA analysis, sequencing, defined as a procedure used to determine the order of the base pairs that comprise DNA, is accomplished by acquiring the sequences of hypervariable regions from a mtDNA sample. MtDNA was put into forensic investigations and was admitted as evidence in a US court in 1996 in the case of State of Tennessee v. Paul Ware. MtDNA was then used to link the two hairs obtained from the scene of crime to the defendant. MtDNA reference samples are also utilized to determine the maternal lineage of a person. In the fall of 1979, a 61-year-old patient wandered away from a US department of Veterans Affairs medical facility and an extensive search was being conducted to recover the missing person. It was after a duration of ten years when a dog discover a human skull in a wooded area near the facility and was believed to be the skull of the patient as mitochondrial DNA profile taken from the patient’s brother matches of the mtDNA found in the skull. Source: FBI Law Enforcement Bulletin, 78 (2002), 21.
Combined DNA Index System (CODIS)
CODIS is a computer software program developed by FBI that maintains local, state and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence and profiles of missing persons. It is used to compare the DNA profiles found on the crime scene and to match against the DNA of a suspect for investigation purposes in the States.
