Genetic Engineering and Human Health

DNA in Forensic Science

Today, approximately half of all criminal cases involve DNA testing. Cases involving paternity, sexual and violent crimes, bioterrorism, disease causing pathogens, and even illegal hunting can involve DNA tests. ¹³ It is well known that DNA makes each person unique. But how and what characteristic of DNA distinguishes one person from another? If twins are essentially identical, what can be used to distinguish them? To answer these questions, an understanding of protein alleles, polymorphisms of DNA, and probability is necessary.

Of the entire DNA in humans approximately 99% are shared between all other humans, leaving our "uniqueness" to occupy 1% of our entire DNA. ¹⁴ To grasp this concept we will need to understand how and where our DNA is unique. For example, there are many variations of the gene for eye color, each variation is called an allele. The terms heterozygous and homozygous describe whether an individual carries two similar pairs of an allele or two different pairs. Alleles come in pairs because genes are inherited, thus one allele is maternally inherited and the other paternally inherited. The actual genotype (genomic makeup) can be determined using Mendel's laws of inheritance. How the genotype is expressed is known as the phenotype. The mixing of genotypes from two parents to a child contributes to the uniqueness of an individual.

Restriction Enzymes

Knowing the genotype for an allele and its possible phenotype and location (locus) has many uses: gene therapy, genetic analysis, and DNA Profiling. A person's DNA profile or DNA makeup can be analyzed with restriction maps. Restriction maps are created from fragments of DNA which have been cut by restriction enzymes. We refer to restriction enzymes as molecular scissors since they are able to recognize specific segment of bases on a strand of DNA. For example, EcoRI is a restriction enzyme that recognizes the sequence 5' – GAATTC- 3'. When the strand is cut between the guanine (G) and the adenine (A), the result is a sticky end DNA fragment. Other restriction enzymes cut DNA in a blunt fashion, with no single strands of bases sticking out. Having some knowledge of restriction enzymes and their target genetic sequence allows us to use probability to predict how long a fragment may be. Since there are only 4 bases, we can predict that the same genetic sequence will occur at every 4 ⁿ base pair, where n is the number of bases recognized. For EcoRI, which recognizes the hexanucleotide of GAATTC, the same sequence would appear every 4 ⁶ or 4096 bp. D. Nicholl in Genetic Engineering states, "fragment length is dependent on the frequency of occurrence of the recognition sequence." ¹⁵ The use of restriction enzymes led to the beginning of a genetic fingerprinting since restriction maps provided a visual representation of a person's genetic makeup, in the form of the lengths of fragments that are produced with a particular restriction enzyme (or combination of enzymes). When comparing two samples of DNA, fragments from two different unrelated persons should show a banding pattern that was different or non-overlapping. On the other hand, two people who are related may show an abundant amount of overlap between their DNA fragment lengths, reflecting similarilities in the base sequences in the DNA. Knowing DNA can be fragmented and targeted paved the way for other studies using restriction maps.

Polymorphisms

With the completion of the Human Genome Project in 2003 geneticists were able to retrieve the exact sequence of human DNA. The location of alleles became known. With this information variations of the gene could be mapped out. Variations in a gene, or polymorphisms, are categorized into two subgroups: Single base polymorphism and length polymorphism. ¹⁶ In a single base polymorphism one complementary pair of bases is replaced with another (i.e. G-C would be replaced with A-T). In the second type or polymorphism, called length polymorphism, a gene sequence has short repeated sequences which extend the length of the strand. For example, a section of DNA may have a region with TAT, but its other form may have 3, 4, 5 repeated TAT in a row rather than one. This in effect lengthens the sequence. ¹⁷

Repeat units of DNA called satellite DNA vary in length from several hundred to several thousand bases in the repeat. Minisatellites or variable number of tandem repeats (VNTRs) are about 10-100 base pairs long. Those that are smaller in length, 2-6 bp, are called microsatellite or short tandem repeats. STRs are a type of length polymorphism that appears in tandem or next to each other. The length of many STRs is only 2-5 bp long but repeated many times in a row. The number of repeats can be distinctive for an individual: using several satellites allows forensic analyst to decrease the chances that a set of STRs belong to someone other than the source of the sample. Thus, the number of STRs and its location on the chromosome contribute to the "uniqueness" of an individual. ¹⁸

Though STR loci mapping is the most widely used technique in forensic DNA testing, there is also an emerging method that has advantages over traditional STR analysis. SNP (Single Nucleotide Polymorphism) genotyping makes use of the abundance of SNPs in the human genome, which is close to 5 million. According to Butler, Coble, and Valone in their paper for Forensic Science Medical Pathology there are several advantages with SNP genotyping. An advantage with SNP use in forensic science is that minute amounts of DNA are needed for SNP analysis. Also highly degraded samples can be analyzed as long as they are amplified. More importantly, a smaller region can be detected since SNPs markers can detect a single nucleotide versus STRs which require many nucleotides. SNPs, unlike STRs, have a relatively low rate of mutation as compared to STRs which have 100 times greater mutation rate. ¹⁹ However, despite the advantages of SNPs, the use of a STR profile in the forensics analysis will not be replaced soon. Butler, Coble, and Valone contend that STRs will continue to be used in the field because an STR profile requires only 13-15 STRs instead of the 40-60 SNPs needed to provide STRs statistical power of a unique match. ²⁰

In recent years, the US has collected and stored DNA information to create DNA banks. One such database is the FBI's CODIS (Combined DNA Index System). CODIS focuses on 13 core STR loci that are used for DNA analysis. Law enforcement officers can compare their samples with known reference samples from CODIS when they lack a suspect. With more profiles added to CODIS many cases, recent and cold, have a higher chance of being solved. Today, many states collect the DNA information from those convicted of violent and non-violent crimes. ²¹

Despite advances in PCR (Polymerase Chain Reaction) technology, DNA collection and handling protocols, errors can arise. To reduce DNA mismatch, experts rarely state that there is a 100% DNA match, often they use the term Random Match Probability (RMP) to describe the uniqueness of DNA profile. This number makes use of the product rule of probability to state the chance of finding the genetic markers within the DNA profile in a target population. The more genetic markers available, the RMP becomes smaller and smaller, increasing the likelihood that the profile is unique to its source. ²²