11.1: Nature of DNA

• Deoxyribonucleic acid (DNA) — is a complex molecule found in nearly all cells in all animals and plants.

• Polymer – a molecule made up of repeating simpler units, called monomers.

  • DNA is located in two regions in a cell, the nucleus, and mitochondria.

  • Nuclear DNA has a geometric shape called a double helix.

  • Helix – a spiral-shaped geometric figure

• Four bases or nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T).

• The order of the base pairs comprises a blueprint or genetic code that determines many of the characteristics of a person.

• The DNA is arranged into 46 structures called chromosomes.

  • The chromosomes are arranged in 23 pairs.

  • One member of each pair of chromosomes comes from the father’s sperm cells, and the other member comes from the mother’s egg cells.

• Male sperm contains 23 chromosomes, and the female ovum (egg) also contains 23.

  • When a sperm and an egg unite, the 23 chromosomes from the sperm and the egg pair up, forming the 46 found in every nucleated cell in the offspring.

• For females, both chromosomes are of the “X” type.

• In males, one of the chromosomes is X and the other is Y.

11.2: Genes and Genetic Codes

• Genes –  sections of chromosomes that function in inheritance and the development of an embryo after conception.

• Homozygous – it is if a person inherits the same form of a gene from the mother and the father.

• Heterozygous – it is if the person receives different forms of the same gene (A and B).

• Allele – each form of a particular gene at a particular locus in the genome.

  • Some alleles are dominant and some are recessive.

• Phenotype – observed characteristic expressed by the gene.

• Genotype – the alleles that make up that gene.

Two types of variability in alleles

1. Sequence polymorphisms

   

2. Length polymorphism

   1. Sample:

      1. Four Score and Seven Years Ago

         Four Score and and Seven Years Ago

         Four Score and and and and Seven Years Ago

         Four Score and and and and and and Seven Years Ago

• Tandem Repeats – the repeats are right next to each other, without any intervening base pairs.

  

• Variable number of tandem repeats – when variation in the number of repeats occurs from one individual to the next.

• Population Genetics – particular genetic markers occur in a given population.

11.3: DNA Typing

• Restriction fragment length polymorphism (RFLP) – the first DNA typing method to be widely adopted by forensic biologists in the analysis of crime.

  • It is not used longer in forensic applications, having been supplanted by methods with higher powers of discrimination and whose results can be obtained faster using much less biological material.

• Polymerase Chain Reaction (PCR) – a technique that is used primarily to increase the amount of DNA by amplification.

• Short Tandem Repeats (STRs) – combine some of the attributes of both PCR and RFLP.

• In RFLP, DNA is extracted from biological material and then restriction enzymes are used to isolate small fragments of DNA called minisatellites or variable number tandem repeats (VNTRs) using restriction enzymes.

  • Probe Hybridization — used to visualize the VNTRs.

11.4: The Polymerase Chain Reaction

• Polymerase chain reaction (PCR) – had been used since the 1970s for making copies (amplifying) of DNA using polymerase enzymes.

The PCR process involves three steps. They all take place within a thermal cycler.

1. Denaturation:

   1. The DNA is added to the PCR tube that contains the reaction mixture and is then heated to 94 °C.

   2. Under these conditions, the double-stranded DNA denatures.

   3. The bonds between the base pairs that hold the strands together break, resulting in single-stranded DNA.

   4. As long as this temperature is maintained, the strands will remain apart.

   5. Each strand will be the template for the formation of a new piece of double-stranded DNA.

2. Annealing:

   1. The next step in the PCR process is to attach a short strand of synthetic DNA to each of the separated strands. These are called primers because they will mark the starting points for the addition of new bases to complete the reproduction of each strand.

   2. The thermal cycler temperature drops to 60 °C for this step.

3. Extension:

   1. The temperature of the reaction is raised to 72 °C.

   2. Under the influence of Taq polymerase, single bases (nucleotides) are added to the primer, one by one.

   3. Each base is complementary to a single nucleotide present on the strand being duplicated.

   4. In this way, the entire complementary strand is built up and a new piece of double-stranded DNA is produced.

   5. This process occurs at each of the complementary single strands created by the denaturation process, so the result is that two identical pieces of double-stranded DNA are produced. This completes one PCR cycle.

   6. The temperature is raised once again to 94 °C and the process repeats.

• Human Leukocyte Antigen (HLA) – first DNA region that was widely subjected to amplification and typing for forensic purposes by PCR.

• DQ Alpha – This gene exhibits sequence polymorphisms.

  • DQ alpha and several other genes collectively called poly markers are typed using a method called reverse dot blot.-

  • Reverse dot blot – the process involves identifying the particular alleles present by reacting them with color-forming reagents on specially treated nylon strips.

11.5: Short Tandem Repeats

• Short Tandem Repeats have the same basic arrangement as VNTRs in that they contain repeating units of base pair sequences in tandem.

  • STRs have certain advantages over VNTRs that make them attractive for forensic comparison purposes.

  • STR markers exhibit high variability in a population, thus giving rise to high degrees of association of evidence with a suspect.

11.6: Mitochondrial DNA

Mitochondria are small structures located within practically all animal and plant cells.

• They function as energy mediators of the cell.

• They take energy released by the metabolism of sugars, fats, and proteins and store it in energetic molecules such as adenosine triphosphate (ATP) which can be used to drive virtually all cell processes.

Differences between mtDNA and genomic DNA

1. mtDNA is circular, containing 26,569 base pairs, and codes for 37 genes.

2. Cells contain many mitochondria, and each one has up to 10 copies of mtDNA. Thus, each cell contains hundreds or thousands of copies of mtDNA, whereas there are only two copies of nuclear (genomic) DNA.

3. mtDNA contains a noncoding region of 1100 base pairs that, in turn, contains two hypervariable regions.

4. All male and female mtDNA comes from the mother. There is no mtDNA from one’s father

5. mtDNA often shows a high degree of variation between unrelated people, making it a powerful tool in forensic typing, however, because there are only two hypervariable regions in mtDNA, the population statistics are not nearly as discriminating as with nuclear DNA.

6. Because of the large number of copies per cell, mtDNA can be useful in typing samples that have low quantities of DNA or in exhibits that are degraded or very old.

The goal of all forensic analysis is to associate a piece of evidence with as few people or objects as possible, and ideally for there to be only one possible source.

• Genetic Concordance –  the relationship between two samples of DNA.

11.7: Interpretation of DNA Typing Results: Purity Issues

• If biological material from a crime scene is relatively clean, fresh, non-degraded, and from only one individual, then the interpretation of the results of DNA typing will usually be straightforward.

• If care is not used in DNA extractions and typing, then biological material from an extraneous source, such as the evidence technician, scientist, or laboratory technician, can be introduced accidentally.

• DNA is a remarkably stable substance, but it can degrade from several causes. These include strong ultraviolet light around 260 nm, humidity, strongly acidic conditions, and oxidizing agents such as strong bleaches and hydrogen peroxide. When DNA degrades, long strands may become fragmented. This fragmentation process is exacerbated by exposure to extreme conditions for longer periods.

11.8: DNA Database: The FBI CODIS System

• Combined DNA Index System (CODIS) – the generic term used to describe the FBI’s program of support for criminal justice DNA databases, as well as the software used to run these databases.

The CODIS system consists of types of three databases, categorized by the type of information they contain.

1. The first contains DNA profiles that are obtained from crime scenes (the forensic database).

2. The first contains DNA profiles that are obtained from crime scenes (the forensic database).

3. The third and most recent database in CODIS is that of missing persons.

Three Levels to the CODIS database system:

1. Local

2. State

3. National Levels