Process involved in DNA Testing




Short Tandem Repeats:  Dinucleotide repeat polymorphisms (1), and trinucleotide and tetranucleotide repeats (2-6), are called Short Tandem Repeats (STRs).  The tandemly repeated consensus sequences are only two to four bases long. The shorter repeat lengths of STR markers make them more compatible with use of the polymerase chain reaction (PCR).  This advantage has made them popular and useful markers for recent genetic maps (7-8).

The application of STR markers to the forensic sciences and paternity analyses requires only a limited number of markers from the thousands which have been generated for genetic mapping purposes.  However, the selected markers must have several characteristics to be useful for human identification.  First, only STRs which demonstrate a high degree of variability within the population should be selected.  Second, the amplified products must be easily distinguished from one another. This means rejecting markers which contain frequent microvariants (i.e., alleles differing from one another by lengths shorter than the repeat length) as the closer and more random spacing of alleles is more difficult to interpret. Finally, the prevalence of stutter bands [i.e., amplification artifacts which appear one or more repeat lengths above or below the true amplified allele (1,9-10)], has led to the rejection of dinucleotide repeats as a class for these applications.

For forensic applications, the ability to amplify and detect very small amounts of DNA template (typically 1 ng) is essential.  For paternity analyses, the mutation rate of the markers must be extremely low to avoid false exclusion of suspected fathers.  In all cases, the reliability and reproducibility of the data is of utmost importance.  In a mapping project, a mistake may mean that a computer program will identify an unlikely double-recombinant which can be reviewed and corrected.  However, the life of one or more individuals will be altered by the outcome of forensic or paternity analyses.

Identification of the best markers for these applications is complicated by the fact that their desired traits are not fully compatible with one another.  While it is possible to identify highly polymorphic markers with a relatively low presence of stutter bands (11-12), such markers generally display microvariants and increased mutation frequency (13-14). 

STRs are similar to VNTRs and the general principles for using them are the same. They differ from VNTRs in having smaller repeat units, from 2 to 7 bases, and the total size of a STR is smaller, usually less than 500 bases.  The smaller size means that the PCR can be used to amplify very small amounts, less than 1 ng, of DNA. It also permits analysis of degraded DNA, that is DNA that is broken into short pieces. Such degraded DNA often cannot be analyzed by Southern blot analysis of VNTRs, which requires higher quality DNA (e.g., larger fragments).  The use of the PCR permits a very tiny amount of DNA, such as would be found on a postage stamp, cigarette butt, or coffee cup, to be amplified to produce an amount large enough to be analyzed (See our forensic cases section).  The PCR also consumes less sample, preserving more material for repeat or referee analysis

The amplified products are separated by electrophoresis as described for the VNTR fragments. But, whereas for VNTRs all the DNA in the cells is on the gel, with STRs only the region of interest is amplified.  However, the smaller range of fragment sizes of STRs and the use of more discriminating separation systems allow identification of all alleles at a locus.  Thus, the requirement for bins is eliminated. 

One allele has an eight repeat the other allele has a seven repeat.

There are, however, micro-variants (i.e., alleles that differ from other alleles by less than one repeat length) which increase discriminating power of the system, but can create problems of band resolution.  In forensic applications, amplified and separated STR fragments are generally detected using one of two methods.  One method uses the propensity of silver to bind to DNA.  The entire gel is stained with silver, but because of the greater amount of DNA in amplified fragments, they stand out against the more dilute background.  A second, increasingly prevalent method requires that some of the primers used during the amplification contain fluorescent tags which are incorporated into the STR fragments generated during amplification. Following fragment separation by electrophoresis, an instrument is used to detect the position of separated fluorescent products. Increasingly, laboratories are using real-time detection during electrophoresis. As in the case of the VNTR systems, the sizes (i.e., number of DNA bases) of the STR fragments detected are used to characterize the sample DNA.  The allele designation for each locus is generally the number of times a repeated unit is present within the identified fragments.  STR loci that have been selected for forensic uses generally have 7 to 30 different alleles.  The population heterozygosity is about 80 percent compared to 90 percent or more for VNTRs.  This relatively small number of alleles compared to the VNTR loci usually leads to unambiguous results, but limits the amount of statistical information and significance that can be obtained from an individual locus.  STR loci are very numerous in the genome and many appropriate loci have already been identified.  Fortunately, it is possible to analyze a DNA sample at many STR loci simultaneously. Such systems (multiplexes) have been developed that allow amplification of 3 to 16 loci at once.  Many forensic laboratories now have instruments that distinguish different fluorescent dyes used to tag particular loci.  These advances have allowed the development of multiplex systems that maintain small amplification product sizes, and therefore can use existing separation methods.

Advantages of STR systems: We selected STR systems for development as genetic markers for forensic science, paternity analysis and tissue culture verification because they offer several advantages over previously employed methods. STR markers are plentiful -- more than two thousand STRs suitable for genetic mapping studies have been described (7-8). From these, we have selected those STRs which have high discrimination potential yet minimal genetic artifacts (such as microvariants) and minimal amplification artifacts (such as stutter bands).

Tandemly repeated DNA sequences are widespread throughout the human genome and show sufficient variability among individuals in a population that they have become important in several fields including genetic mapping, linkage analysis, and human identity testing.  These tandemly repeated regions of DNA are typically classified into several groups depending on the size of the repeat region.  Minisatellites (variable number of tandem repeats, VNTRs) have core repeats with 9-80 bp, while microsatellites (short tandem repeats, STRs) contain 2-5 bp repeats.  The forensic DNA community has moved primarily towards tetranucleotide repeats, which may be amplified using the PCR with greater fidelity than dinucleotide repeats.  The variety of alleles present in a population is such that a high degree of discrimination among individuals in the population may be obtained when multiple STR loci are examined.

Advantages of STRs over traditional RFLP techniques: PCR-based STRs have several advantages over conventional Southern blotting techniques of the larger variable number tandem repeats (VNTRs). Discrete alleles from STR systems may be obtained due to their smaller size, which puts them in the size range where DNA fragments differing by a single basepair in size may be differentiated.  Determination of discrete alleles allows results to be compared easily between laboratories without binning. In addition, smaller quantities of DNA, including degraded DNA, may be typed using STRs. Thus, the quantity and integrity of the DNA sample is less of an issue with PCR-based typing methods than with conventional RFLP methods.

The advantages of STRs are:

1. The process can be used with degraded samples (since shorter fragments of DNA can be analyzed).

2. The PCR process permits analysis of extremely small amounts of DNA.

3. The potential number of loci is very large. This is particularly important if siblings or other relatives are involved.

4. The process is rapid; it may be completed in a day or two.

5. The system lends itself to multiplexing and automation.

STR loci commonly used in DNA typing:  There are literally hundreds of STR systems which have been mapped throughout the human genome. Several dozen have been investigated for application to human identity testing {17-19}.  These STR loci are found on almost every chromosome in the genome. They may be amplified using a variety of PCR primers.  Tetranucleotide repeats have been most popular among forensic scientists due to their fidelity in PCR amplification although some tri- and pentanucleotide repeats are also in use (15-19).

Desirable features for STR systems include

  • high heterozygosity
  • regular repeat unit
  • distinguishable alleles
  • robust amplification

References:  1. Weber J.L. and May, P.E. (1989) Am. J. Hum. Genet. 44, 388; 2. Edwards, A. et al. (1991) In: The Second International Symposium on Human Identification, Promega Corporation, 31; 3. Edwards, A. et al. (1991) Am. J. Hum. Genet. 49, 746; 4. Polymeropoulos, M.H. et al. (1991) Nucl. Acids Res. 19, 4018; 5. Polymeropoulos, M.H. et al. (1991) Nucl. Acids Res. 19, 4306; 6. Polymeropoulos, M.H. et al. (1991) Nucl. Acids Res. 19, 3753; 7. Murray, J.C. et al. (1994) Science 265, 2049; 8. Adamson, D. et al. (1995) Am. J. Hum. Genet. 57, 619; 9. Levinson, G. and Gutman, G.A. (1987) Mol. Biol. Evol. 4, 203; 10. Schlotterer, C. and Tautz, D. (1992) Nucl. Acids Res. 20, 211; 11. Sprecher, C.J. et al. (1996) BioTechniques 20, 266; 12. Lins, A.M. et al. (1996) BioTechniques 20, 882; 13. Moller, A., Meyer, E. and Brinkmann, B. (1994) Int. J. Leg. Med. 106, 319; 14. Brinkmann, B., Moller, A. and Wiegand, P. (1995) Int. J. Leg. Med. 107, 201. 15. Wyman, A.R. and White, R. (1980) Proc. Natl. Acad. Sci. USA 77, 6754; 16. Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Nature 316, 76; 17. Donis-Keller, H. et al. (1987) Cell 51, 319; 18. Schumm, J.W. et al. (1988) Am. J. Hum. Genet. 42, 143; 19. Nakamura, Y. et al. (1987) Science 235, 1616.

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