Visualization of VNTRs
Once the DNA has been transferred to the nylon membrane, a technique called probe hybridization is used to visualize the VNTRs. When RFLP was first developed for forensic use, short strands of DNA that are complementary to the core-repeating units of the VNTRs, were radioactively labeled. These short sequences of single-stranded DNA (probes) were given the opportunity to bind (hybridize) to complementary single-stranded sequence on the membrane. When the membrane was brought in contact with photographic or X-ray film, the labeled fragments exposed the film, creating an autoradiogram or autorad. In recent years, radioactive labeling has given way to the use of chemiluminescence, whereby the probes are labeled with a chemical that will react with a chemiluminescent substrate to produce visual VNTRs. Figure 1 illustrates probe hybridization using a radioactive label.
With the help of the calibration ladder, the lengths of each of the VNTRs can be determined. As RFLP was developed, the more number of VNTRs were isolated and analyzed and more data were collected that determined the population frequencies of occurrence of each band of each VNTR. One of the limitations of RFLP typing is that it is not possible to resolve a band into an exact VNTR length. A band con sists of a small group of fragments whose lengths differ by a few base pairs. Thus, VNTR fragments cannot be viewed as discrete alleles. Because of this, scientists have been conservative in interpreting RFLP results. Instead of treating a fragment as a unique allele, they are put into bins of various sizes that take measurement errors into account. When an RFLP was run on a case and a match occurred between a known and unknown DNA sample, the probability that this match was a coincidence could be determined.
FIGURE 1 The probe hybridization process. After the DNA fragments have been separated, they are single–stranded. Special probes, short sequences of DNA that are complementary to the VNTR’s are put onto the membrane and they will attach to their complementary sequences. These probes are labeled either radioactively or by chemiluminescence so that they can be easily visualized.
