Opinion ID: 883138
Heading Depth: 1
Heading Rank: 5

Heading: RFLP Analysis

Text: As is explained in Commonwealth v. Curnin, 565 N.E.2d at 446-47, and U.S. v. Jakobetz, 955 F.2d at 792-93, RFLP analysis involves several steps. 1. Extraction of DNA. The DNA must be extracted from the evidentiary sample by using chemical enzymes. An enzyme is then added to digest cellular material that is not DNA, thereby providing a purer sample. 2. Restriction or Digestion. The DNA is then mixed with restriction enzymes which cut the DNA molecules into fragments at specific base sequences. The restriction enzymes recognize particular sequences of base pairs. The enzymes sever the DNA molecule at targeted locations within the sequence. The process severs the DNA molecule at all sites targeted at locations along the three billion base pair length of the molecule. Therefore, some of the resulting restriction fragments will contain polymorphic DNA segments, although most will not. Because the alleles differ markedly in length from one person to the next, the restriction fragments containing the alleles will also differ in length. 3. Gel Electrophoresis. This technique entails placing the DNA fragments into an agarose gel which has a negative and positive electrode at either end. An electrical current is then run through the gel. The restriction fragments, which are negatively charged in their natural state, travel toward the positive charge. The process is able to sort the restriction enzymes by length, as the shorter fragments  which are lighter and less bulky  will travel further in the gel. Several samples are run on the gel but in different tracks or lanes which run parallel to each other. In addition to the samples, fragments of known base-pair lengths are placed in separate lanes to facilitate measurement. 4. Southern Transfer. This procedure transfers the fragments to a more functional surface. Anylon membrane is placed over the gel and, through capillary action, the DNA fragments attach themselves to the membrane while occupying the same position relative to one another as they had on the gel. The restriction fragments are then treated with a chemical which cuts the fragments of DNA lengthwise along each base pair, by sawing through the middle of each rung. The result is a collection of single stranded restriction fragments. 5. Hybridization. The nylon membrane is dipped into a solution containing various genetic probes, which are single stranded DNA fragments of known length and sequence designed to link with identified polymorphic alleles. The probes will link only to those DNA fragments which contain base pair sequences that are complementary to the base sequences of the probe. The genetic probes are tagged with a radioactive marker so that after the probe links with a particular allele, its position relative to the other restriction fragments can be observed. 6. Autoradiography. The nylon membrane is placed on an x-ray film and exposed by the radioactively charged probes. The result is a pattern of bands called an autoradiograph, or autorad. Each band represents a different polymorphic allele, and its position indicates the length of the restriction fragment in which that allele occurs. Because individuals differ in length of their polymorphic alleles, the position of the bands on the DNA prints will tend to differ from person to person. 7. Interpretation of the DNA Print. The DNA print of the crime sample and the DNA print of the defendant are then compared both visually and with a machine to determine if both samples of DNA came from the same person. A match will be declared if the samples fall within a certain distance of one another. Cellmark Diagnostics, the laboratory conducting the RFLP analysis in this case, will declare a match if the bands from two DNA prints fall within one millimeter of each other. 8. Statistical Analysis. Statistical analysis is used in both RFLP and PCR analysis. If the two DNA samples match, then population geneticists determine the likelihood that the match is unique. The scientists determine the frequency with which a particular allele is found in the population, then by using a multiplication or product rule, compute an aggregate estimate of the statistical probability that the suspect's combination of alleles would be found in the relevant racial population. United States v. Bond (6th Cir.1993), 12 F.3d 540, 550, aff'g United States v. Yee, (Ohio 1991), 134 F.R.D. 161.