FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
SUBJECT FORENSIC SCIENCE
Paper No. and Title PAPER No.13: DNA Forensics Module No. and Title MODULE No.29: DNA Typing
Module Tag FSC_P13_M29
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing TABLE OF CONTENTS
1. Learning Outcomes 2. Introduction
3. RFLP typing using multi locus and single locus probes.
4. PCR-based typing- Reverse slot blot, Amp-FLP, STR typing, mtDNA sequencing
& SNP typing 5. Summary
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing 1. Learning Outcomes
After studying this module, the readers shall be able to understand -
The Forensic DNA Typing Techniques developed so far since 1984
The steps involved in techniques and their limitations
The interpretation of the DNA Typing results
2. Introduction
Forensic DNA Typing also referred to as “DNA Fingerprinting” or “DNA analysis” or “DNA profiling “(as we know it today) is the most significant advancement for use in justice administration in civil and criminal cases. The discovery of highly polymorphic regions in human DNA by Wyman and White (1980) laid the foundation for the discovery of individual specific banding pattern observed after restriction fragment length polymorphism analysis of repeated DNA sequences by Professor Sir Alec Jeffrey at the University of Leicester. He found certain regions of DNA containing DNA sequences which were repeated over and over again next to each other. He also observed the number of repeated sections present in a sample could differ from individual to individual. These repeat regions were known as VNTRs, or Variable Number of Tandem Repeats. The technique used by him to examine the VNTRs was called Restriction Fragment Length Polymorphism (RFLP) as it involved use of restriction enzymes for cutting the regions of DNA surrounding the VNTRs.
This breakthrough in DNA “fingerprinting” focused the attention of forensic scientists of Home Office Forensic Science Service, UK and the procedure for extraction of DNA from dried stains of human body fluids were developed. Subsequently, commercial introduction of the PCR process in 1987 accelerated the pace and widened the scope of application of DNA technology in the forensic field. Several commercial laboratories like Cellmark Diagnostics in UK, Lifecords Corporations & Cetus Corporations of USA played significant role in developing and standardizing various techniques and protocols.
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
After extraction of DNA from the target tissues, its purification and assessment of its integrity, earlier RFLP method was used for DNA typing which was subsequently replaced by PCR-based methods. RFLP method encompasses fragmenting the DNA strands into desired lengths using restriction enzymes, separating the DNA fragments through the gel medium according to their sizes (molecular weights), transferring the fragments on to the solid supports (nylon or cellulose membrane) hybridizing the specific DNA fragments with complementary DNA pieces (probes tagged with radioactive or non-radioactive material) and taking the images or impressions of the desired fragments.
PCR-based methods involve amplification of a small or specific portion of DNA or its loci for further manipulation for desired goal (Amp-FLP, STRs, Mini-STRs, mtDNA and SNPs).
Fig. 1: Overview of the forensic DNA typing techniques
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
3. RFLP typing using multi locus(MLP) and single locus (SLP) probes
Sets of base sequences repeated numerous times are present within the human genome. These repeated sequences are called mini-satellites which show a very high degree of allelic variation in the number of repeat units and accordingly in their length. Hence, they form the basis of differentiating or identifying individuals based on this length polymorphism at molecular level by using either multi-locus or single locus probes.
Use of only one multi-locus probe can provide sufficient numbers of variable bands which establish positive identity of an individual. Thus, it is a single powerful test for positive matching of body tissues and determination of parentage. Probability of obtaining identical patterns of bands from the DNA of two individuals using multi-locus probe is of the order of 1 in 1014 to 1030 which is more than 5X109, the total world population. Thus, the DNA prints obtained from multi-locus probes are highly unique.
For detecting variation at specific mini-satellite loci single locus probe or mixture of two or more probes were used. A single locus probe reveals a pattern of up to two bands whereas mixture of several probes reveals more than 2 bands. These probes are highly sensitive, and hence can be used for small and even partially degraded samples of DNA. These are useful in identification of mixed samples as in cases of multiple rapes.
The RFLP DNA testing has 4 basic steps.
a) Restriction digestion: After isolation of DNA from any biological sample the specific quantity of DNA sample is treated with restriction endonucleases (Hae 111, Hind 111, Hinf 1, Pst 1 etc.).This step is called restriction digestion of the target DNA sample. The endonucleases are capable of recognizing and cutting the DNA at specific sites along its sequence resulting into a reproducible set of restriction fragments of various lengths. The restriction enzyme cut the DNA into thousands of small fragments of nearly all possible sizes.
b) Separation of restriction fragments: Fragment of DNA are separated according to sizes on agarose gel under the influence of electric current (electrophoresis) wherein the gel acts like a sieve through which small DNA fragments move through the pores of gel rapidly as compared to larger fragments.
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
c) Southern blotting: The separated fragments are visualised by Southern blotting where membrane are transferred from the gel onto nylon paper (capillary transfer or vacuum transfer).
d) Hybridization: The DNA fragment fixed on to nylon membrane is hybridized (bound) with either multi-locus probe (MLP) or single locus probe (SLP). In SLP, each probe is specific for a single trait or single locus in the genome. These probes are DNA sequence, labelled with radioactive or chemiluminescent substances. MLPs consist of tandem repeats containing a mini-satellite “core” sequence capable of simultaneously detecting a number of highly polymorphic loci having same “core”
sequence and thus generating individual-specific DNA fingerprints. If radioactively labelled probe is used, the nylon membrane/blot is laid on to x ray film. Bands are formed on the x ray film on the site of radioactive substance present on the blot. This x ray film is called “autorad”. Alternatively, the bands are visualized using chemiluminescent dyes.
Fig. 2: RFLP-MLP in first immigration case
First application of DNA fingerprinting in which two MLPs were used to analyse samples for immigration case. In this case sample of mother, her disputed male child, three undisputed children and one unrelated individual were analysed by Jeffery.
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
Fig. 3: RFLP-MLP in rape and disputed paternity
Fig. 4: First application of DNA profiling in a crime case using SLP
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
4. PCR-based typing- Reverse slot blot, Amp-FLP, STR typing, mtDNA sequencing & SNP typing
Some VNTR loci have relatively short size alleles. These loci can be PCR amplified. Locus D1S80 was used in forensic setup for Amp-FLP analysis. The fragments in this locus are in range of 14 to 42 repeat units (16bp per unit). The amplified fragments were separated according to size using polyacrylamide gel electrophoresis and detected using silver stain.
The discrete alleles are compared directly with the allelic ladder. The Amp-FLP technique requires less DNA than RFLP method and worked well for degraded samples. This locus can be analysed in multiplex fashion with amelogenin locus for gender typing.
Fig. 5: Amp-FLP locus D1S80 amplified by PCR
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
Short Tandem Repeat (STR) a sub-category of VNTR, is a region of human DNA containing 2 to 7 base pairs (bps) repeat unit. These STRs are also called microsatellite or simple sequence repeats. More than 105 STRs exist in human genome out of which some STRs have been characterized specifically for forensic DNA profiling. Earlier the STR profiling was carried out using poly acrylamide gel electrophoresis of the PCR product and staining the gel with silver stain.
Fig. 6: Gel plates showing separation of alleles of three STR loci on each
Fig.6 shows the STR typing at 9 loci (three on each plate) where sample number 1, 2, 3 and 4 display the allele which is given number against the allele in the ladder (L) run along with the sample in the gel. Presently, STR profiling is being done using multiplex PCR system in which multiple fluorescent dyes are used to detect the amplified product using capillary electrophoresis in automatic DNA sequencer.
Sequence polymorphism at DQ A1 and 5 other polymorphic loci (LDLR, GYPA, HBGG, D7S8, and GC) on human DNA were analysed based on the phenomena of complimentary base pairing. Under appropriate condition only those single strands which match exactly will hybridize. If only one or a few bases are different, the two strands will fail to attach to each other.
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
Commercially available nylon strips to which DNA probes are attached are treated with PCR products. Finding complimentary sequence, the target DNA fragments in PCR products bind to the strip at the location of the probe. The primers (probe) used in the reaction are labelled with biological tag called biotin. On reacting with a protein streptavidin/horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) with hydrogen peroxide blue colour dots appear at the site of binding of DNA sequence with probe. Pattern of dots correspond to alleles present in the sample. The results are recorded and saved by photography.
Fig. 7: Detection of PCR product on reverse dot blot
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
Fig. 8: AmpliType PM (polymarker)
Fig. 9: DNA typing of heterozygous alleles at the HLA-DQA1 locus using the reverse blot assay
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing
mtDNA sequencing is carried out when the samples are old or badly degraded, often bones, teeth and hair or in cases where amount of nuclear DNA is very small for STR profiling. The Sanger method developed in 1977 is still widely used for mtDNA sequencing in which each DNA strand is sequenced in separate reactions with a single primer either forward or reverse.
Four different coloured fluorescent dyes are attached to the four different ddNTPs. These are similar dyes to those used for STR detection. DNA sequencing of mtDNA is carried out in following steps:
1. PCR amplification of the entire control region or a portion of it with various primer sets.
2. Removal of remaining dNTPs and primers from PCR through spin filtration or enzymatic digestion.
3. Determination of PCR quantity.
4. Performance of DNA sequencing reaction to incorporate fluorescent ddNTPs with each reaction containing a different primer to dictate which stand is sequenced.
5. Removal of unincorporated fluorescent dyes terminators from the completed sequencing reaction.
6. Dilution of purified sequencing reaction products in formamide.
7. Separation through capillary electrophoresis instrument.
8. Sequence analysis of each reaction performed.
Single nucleotide polymorphisms (SNPs) are used for human identification, paternity testing, hair colour and eye colour identification in forensic setup. Over the years a number of methods have been developed for SNP analysis such as measurement of fluorescence, luminescence and molecular mass. Most assays are carried out in solutions or on solid matrix support such as glass slide, chip or bead. Autosomal SNPs can be used for many type of forensic testing including analysis of degraded samples. SNP loci on Y chromosome are also potential marker for paternity testing because of low mutation rate.
FORENSIC SCIENCE PAPER No.13: DNA Forensics MODULE No.29: DNA Typing 5. Summary
DNA profiling was developed, due to two independent breakthroughs in molecular biology at the same time in UK and US when Polymerase Chain Reaction (PCR) was developed by Kary Mulis of Cetus Corporation US, and individual- specific banding patterns were observed after Restriction Fragment-Length Polymorphism (RFLP) analysis of repeated DNA sequences by professor sir Alec Jefferys at the university of Leicester UK.
In RFLP technique multi locus and single locus probes are used. This technique is capable of generating highly individual-specific DNA profiles. Since, in this method relatively large amounts of DNA are required, it is less vulnerable to contamination by extraneous sources (e.g. investigating officers or laboratory personnel). However, it suffers from several shortcomings like the limited sensitivity of detection due to which it is difficult or impossible to obtain meaningful profiles from trace biological evidence or from samples that have significantly compromised due to aging or environmental insults. In addition, the interpretation of VNTR profiles also difficult sometimes due to artifacts of the procedure such as partial Hae III digestion, band shifting, and incomplete profiles due to faint hybridization of low-molecular-weight alleles or degradation of high-molecular-weight alleles. RFLP typing is quite time consuming, laborious and difficult to automate.
Limitations of RFLP typing have been overcome through the development of PCR- based DNA typing systems.
