An Overview of Forensic Typic Systems & Procedures for Forensic DNA Analysis

 

Restriction Fragment Length Polymorphisms (RFLPs) - historically significant but not used much anymore

RFLPs can arise in several ways:

Although either of the above situations can lead to polymorphic differences in the population, many of the specific loci that are used in criminal forensic cases and in paternity cases involve repetitions of a core sequence at a given genetic locus. The length of each repeat unit and the number of repeat units varies. Therefore, the length of the fragment generated by a restriction enzyme that cuts on either side of (but not within) the locus also varies. These types loci are referred to as
Variable Number of Tandem Repeats loci (VNTRs).

 

Since the amount of material that is different can vary from individual to individual, these differences take on polymorphic distributions within a population. Furthermore, these polymorphisms are inherited with typical Mendelian patterns that can be used to identify parental lineage as well as individual differences. Therefore, these RFLPs have use in forensic crime scene analysis and in paternity cases. The basic process involves: 

 

 

 

  1. Processing appropriate samples to obtain the DNA
  2. Digesting the DNA with a particular restriction endonuclease (HaeIII has generally been used as the standard)
  3. Separating the resulting DNA fragments by electrophoresis
  4. Transferring the DNA to a membrane (Southern Transfer)
  5. Probing that membrane with one or more specific probes that have been shown to demonstrate specific, heritable, polymorphic allelic differences among individuals in the population It is not uncommon to use as many as 5 different probes in order to generate probabilities that virtually exclude any other individuals.

 

AmpliType PM & DQA1 - Sequence Polymorphisms

The first PCR based DNA Typing systems eventually resulted in the Commercial availability of the AmpliType PM & DQA1 system from Applied Biosystems, INC (ABI; formerly Perkin-Elmer). The loci involved in this system are all sequence polymorphisms that are detected/delineated by hybridization to allele-specific oligonucleotide (ASO) probes. The discriminatory power of this system is much more limited than RFLP analysis (approximately 1:2000) because only a limited number of alleles (and therefore, a limited number of genotypes) exist in the population. Nevertheless, the system is useful when evidence samples yield limited amounts of DNA and/or DNA that is too degraded for RFLP analysis.

The AmpliType PM & DQA1 system tests for polymorphisms at 6 loci:

The basic steps in the procedure are:

  1. Simultaneous PCR amplification of the six loci using primers that have been conjugated to biotin
  2. Hybridization (allele-specific) of the PCR products using a reverse dot-blot method in which the ASO probes have been impregnated onto membrane filter strips in specific locations; two strips are used: one for the DQA1 locus and one for the remaining 5 loci of the Polymarker (PM)
  3. Binding of a horseradish peroxidase/streptavidin conjugate to the hybridized biotinylated PCR product
  4. Colorimetric detection of the hybridized PCR product-biotin/HRP-streptavidin complex using a chromagenic substrate for the HRP

Typical results are shown below:

Polymarker (PM) Reverse Dot Blot

DQA1 Reverse Dot Blot

 

Because of its low Power of Discrimination (Pd) this system is seldom used anymore. However, it is still commercially available and used occasionally

 

D1S80 - Amplified Fragment Length Polymorphism (AFLP)

VNTRs, such as described above for RFLP analysis, can also be detected by Polymerase Chain Reaction (PCR) Amplification. Primers are designed to anneal to sequences in the DNA that flank the VNTR. PCR Amplification of the material between the primer locations includes the VNTR region. Therefore, any allelic differences between individuals will be evidenced by different lengths for the amplification product among individuals tested. The D1S80 locus has been used this way. It has a core repeat unit of 16 bp, which is repeated 14-41 times.

 

The basic process involves: 

  1. Processing appropriate samples to obtain the DNA
  2. PCR Amplification of a region of the DNA containing a polymorphic VNTR(i.e. D1S80)
  3. Separating the resulting DNA amplification products by electrophoresis
  4. Analysis of the resulting pattern by UV transillumination of Ethidium Bromide stained gels or by Silver Staining to determine inclusion or exclusion

Advantages:

Disadvantage:

Typical results are shown below:

 

Short Tandem Repeats (STRs; Microsatellites)

Short Tandem Repeat sequences (STRs) are similar to VNTRs in that they involve tandem repeats of a core sequence in variable numbers among the population to produce a polymorphic distribution. The major difference is that the core sequence is usually only 3 or 4 nucleotides in length (VNTR core sequences can be 16 or more nucleotides). Usually a tighter range of alleles results. The small size of the STRs used in forensic DNA profiling (amplimers range from 100 - 500 bp) allows for more efficient amplification by PCR and also allows the use of DNA that has been degraded more significantly because even small pieces of DNA may contain intact STR sites. Primers are designed to anneal to sequences in the DNA that flank the STR. PCR Amplification of the material between the primer locations includes the STR region. Therefore, any allelic differences between individuals will be evidenced by different lengths for the amplification product among individuals tested.

The basic differences between classical RFLPs, VNTRs and STRs are illustrated in the diagram below:

Schematic diagram illustrating different types of polymorphic markers. RFLP markers are distinguished by polymorphism due to presence or absence of a restriction site at the same site in homologous chromosomes. VNTR and STR markers contain repeated sequences of 15-70 and 2-4 bases, respectively. VNTR markers are detected using flanking restriction sites (as shown) or using PCR primers, in which case they are called "amplifiable VNTRs" or "AMP-FLPs". Unique flanking sequences are used as PCR primers with STR markers.

 

 

Multiplex STRs

PCR Process

 

A number of STR loci have been identified with non-overlapping allelic distributions. This allows the use of multiplex PCR amplification and separation of multiple loci in a single lane of a polyacrylamide gel. The use of different fluorescent dyes allows the multiplex amplification of STR loci that have overlapping allele ranges as long as a separate dye is used for each overlapping STR. The dyes are coupled to the primers used for the amplification so that the different loci can be readily identified. An illustration of one such system, available from PromegaTM is illustrated below. A clearer picture of the overlapping alleles can be obtained by using a filter that masks all but one of the dyes producing a black & white image of the alleles stained with one of the dyes. This is also illustrated in the figure below (reprinted from PromegaTM):

The GenePrint® PowerPlex™ 2.1 System from PromegaTM. Twelve DNA samples (lanes 1­12) were amplified and are shown with allelic ladders (lanes L) for each of the nine loci contained in the GenePrint® PowerPlex™ 2.1 System. Panel A: Three-color display with fluorescein-labeled loci, Penta E, D18S51, D21S11, TH01 and D3S1358, shown in green; carboxy-tetramethylrhodamine-labeled loci, FGA, TPOX, D8S1179 and vWA, shown in red; and the carboxy-X-rhodamine-labeled ILS 600 fragments of the size marker shown in blue. Panel B: Scan using a 505nm filter, which reveals a black and white image of the fluorescein-labeled loci, Penta E, D18S51, D21S11, TH01 and D3S1358. Panel C: Scan using a 585nm filter, which reveals a black and white image of the TMR-labeled loci, FGA, TPOX, D8S1179 and vWA. In Panels B and C, each allelic ladder is labeled to the right with the number of copies of the repeated sequence contained within its corresponding largest and smallest alleles. All materials were separated using a 5% Long Ranger™ denaturing polyacrylamide gel and detected using the Hitachi FMBIO® II Fluorescence Imaging System.

STRs can also be analyzed by Capillary Electrophoresis instead of on a gel. Results below show a tytpical output from the ABI PRISM 310 Genetic Analyzer using the fluorescent labeled Profiler Plus (top panel) and COfiler STR systems (also from ABI). Different colored dyes are used for STRs with overlapping alleles:

 

Gender ID (Amelogenin)

The amelogenin locus is a PCR based gender determining locus. In a kit supplied by Applied BioSystems, the sequence on the X-chromosome amplifies a 103 bp fragment while the corresponding sequence on the Y-chromosome amplifies a 109 bp fragment. Therefore, males (XY) show a heterozygous condition with 2 different sized fragments while females (XX) show a homozygous condition with only the smaller (103 bp) fragment. Alternatively, Promega Corporation supplies a kit with primers that span a larger distance surrounding the polymorphic site so that amplification of the X chromosome sequence produces a 212 bp fragment while amplification of the the Y chromosome region produces a 218 bp fragment.

 

Mitochondrial D-Loop DNA (a sequence polymorphism)

Mitochondrial DNA is inherited in a direct matrilinear manner. It is approximately 16.5 kbp and contains a noncoding, hypervariable region (1,100 bp) called the D-Loop. Two particular sequences within this region tend to mutate with extremely high frequency and thus allow for highly variable sequence polymorphisms among unrelated individuals.

Y-Chromosome STRs

The Y chromosome is inherited in a direct patrilinear manner. Since it is only found in males it can be useful in a variety of situations where a mixture of male & female donors could obscure the results.

 

The Future of DNA Typing Systems

The major problem for the forseeable future is not increasing the Power of Discrimination but rather increasing throughput so that more samples can be processed in a reasonable period of time to ensure speedy trials. New instruments and new methodologies are being developed with this goal in mind. Adoption of new methodologies in the forensic community can be slow because of the need for extensive validation and the development of QA/QC protocols.

For More Information About STRs Go To The STR Database