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The DNA fragments within the blot are permanently fixed to the filter, and the DNA strands are denatured. Radiolabeled probe molecules are then added that are complementary to sequences in the genome that contain repeat sequences. These repeat sequences tend to vary in length among different individuals and are called variable number tandem repeat sequences or VNTRs. The probe molecules hybridize to DNA fragments containing the repeat sequences and excess probe molecules are washed away. The blot is then exposed to an X- ray film. Fragments of DNA that have bound to the probe molecules appear as dark bands on the film. The Southern blot technique is laborious, and requires large amounts of undegraded sample DNA. Also, Karl Brown's original technique looked at many minisatellite loci at the same time, increasing the observed variability, but making it hard to discern individual alleles (and thereby precluding paternity testing). These early techniques have been supplanted by PCR- based assays. PCR analysis. This has revolutionized the whole field of DNA study. The process, the polymerase chain reaction (PCR), mimics the biological process of DNA replication, but confines it to specific DNA sequences of interest. With the invention of the PCR technique, DNA profiling took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA. In the PCR process, the DNA sample is denatured into the separate individual polynucleotide strands through heating. Two oligonucleotide DNA primers are used to hybridize to two corresponding nearby sites on opposite DNA strands in such a fashion that the normal enzymatic extension of the active terminal of each primer (that is, the 3. PCR uses replication enzymes that are tolerant of high temperatures, such as the thermostable Taq polymerase. In this fashion, two new copies of the sequence of interest are generated. Repeated denaturation, hybridization, and extension in this fashion produce an exponentially growing number of copies of the DNA of interest. Instruments that perform thermal cycling are now readily available from commercial sources. This process can produce a million- fold or greater amplification of the desired region in 2 hours or less. Early assays such as the HLA- DQ alphareversedot blot strips grew to be very popular due to their ease of use, and the speed with which a result could be obtained. However, they were not as discriminating as RFLP analysis. It was also difficult to determine a DNA profile for mixed samples, such as a vaginal swab from a sexual assault victim. However, the PCR method was readily adaptable for analyzing VNTR, in particular STR loci. In recent years, research in human DNA quantitation has focused on new . Quantitative PCR methods enable automated, precise, and high- throughput measurements. Interlaboratory studies have demonstrated the importance of human DNA quantitation on achieving reliable interpretation of STR typing and obtaining consistent results across laboratories. STR analysis. This method uses highly polymorphic regions that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases). Because unrelated people almost certainly have different numbers of repeat units, STRs can be used to discriminate between unrelated individuals. These STR loci (locations on a chromosome) are targeted with sequence- specific primers and amplified using PCR. The DNA fragments that result are then separated and detected using electrophoresis. There are two common methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis. Each STR is polymorphic, but the number of alleles is very small. Typically each STR allele will be shared by around 5 - 2. The power of STR analysis comes from looking at multiple STR loci simultaneously. The pattern of alleles can identify an individual quite accurately. Thus STR analysis provides an excellent identification tool. The more STR regions that are tested in an individual the more discriminating the test becomes. From country to country, different STR- based DNA- profiling systems are in use. In North America, systems that amplify the CODIS 1. United Kingdom the DNA- 1. The National DNA Database) is in use. Whichever system is used, many of the STR regions used are the same. These DNA- profiling systems are based on multiplex reactions, whereby many STR regions will be tested at the same time. The true power of STR analysis is in its statistical power of discrimination. Because the 1. 3 loci that are currently used for discrimination in CODIS are independently assorted (having a certain number of repeats at one locus does not change the likelihood of having any number of repeats at any other locus), the product rule for probabilities can be applied. This means that, if someone has the DNA type of ABC, where the three loci were independent, we can say that the probability of having that DNA type is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1x. However, DNA database searches showed much more frequent than expected false DNA profile matches. The risk is greater for matching the most common person in the samples: Everything collected from, or in contact with, a victim is a major source of contamination for any other samples brought into a lab. For that reason, multiple control- samples are typically tested in order to ensure that they stayed clean, when prepared during the same period as the actual test samples. Unexpected matches (or variations) in several control- samples indicates a high probability of contamination for the actual test samples. In a relationship test, the full DNA profiles should differ (except for twins), to prove that a person was not actually matched as being related to their own DNA in another sample. Another technique, Amp. FLP, or amplified fragment length polymorphism was also put into practice during the early 1. This technique was also faster than RFLP analysis and used PCR to amplify DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish various alleles, which were separated on a polyacrylamide gel using an allelic ladder (as opposed to a molecular weight ladder). Bands could be visualized by silver staining the gel. One popular locus for fingerprinting was the D1. S8. 0 locus. As with all PCR based methods, highly degraded DNA or very small amounts of DNA may cause allelic dropout (causing a mistake in thinking a heterozygote is a homozygote) or other stochastic effects. In addition, because the analysis is done on a gel, very high number repeats may bunch together at the top of the gel, making it difficult to resolve. Amp. FLP analysis can be highly automated, and allows for easy creation of phylogenetic trees based on comparing individual samples of DNA. Due to its relatively low cost and ease of set- up and operation, Amp. FLP remains popular in lower income countries. DNA family relationship analysis. The zygote contains a complete set of DNA molecules, a unique combination of DNA from both parents. This zygote divides and multiplies into an embryo and later, a full human being. At each stage of development, all the cells forming the body contain the same DNA. 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