DNA Fingerprinting

DNA Analysis First described by Alec Jeffreys, analysis of DNA from biological material is referred to as DNA fingerprinting. However, the process is misnamed, since DNA analysis does NOT point to a unique individual. Analysis of DNA evidence provides a profile. If a suspect's DNA matches the profile, forensic professionals can calculate the probability that there are others who also match the profile. The term DNA profile is more accurate than DNA fingerprint.

DNA profiling evidence has raw graphic power in the courtroom. A layperson, or juror, can see the matching pattern or the lack of a match for excluded suspects. Results are clear without any knowledge of biology; a match seems to say "The suspect is clearly guilty."

Take a look at all the pieces of the puzzle!

Alleles and Forensic Analysis

  • The genes used in forensic analysis often come in 20 or more different alleles
  • Each individual only has 2 alleles in his/her genome
  • If the 2 alleles are identical, the person is homozygous
  • If the 2 alleles are different, the person is heterozygous

The VNTR

  • One type of multiple allele has a sequence of 20 to 40 bases repeated many times over and over in tandem
  • The number of repeats varies for different individuals in the population
  • Therefore, these alleles are called Variable Number Tandem Repeats (VNTR)

VNTR Loci in Forensics

  • The VNTR loci currently used in forensic identification have been mapped on specific chromosomes
  • Occur only once in the genome
  • Are on separate chromosomes, and thus, inherited independently (product rule)
  • All the evidence to date indicates that they show linkage equilibrium

Restriction Endonucleases

  • Enzymes that cut DNA at specific base sequences known as restriction endonuclease sites
  • By proper choice of a restriction endonuclease, the DNA can be cut to yield fragments that reveal the number of terminal repeats in a VNTR

Two Restriction Endonuclease Cleavage Sites

  • PstI: C T G C A G G A C G T C
  • HaeIII: G G C C C C G G

VNTR Analysis

  • Requires a sufficiently large sample of DNA
  • DNA Profiles are based on sites in the DNA of different individuals with Variable Numbers of Tandem Repeats (VNTR)

PCR Analysis

PCR Analysis

  • When only small amounts of DNA are available for analysis, one can use PCR
  • Polymerase Chain Reaction (PCR) is based on the amplification and identification of specific unique sequences in the DNA which vary among different individuals

How Much DNA Is Enough? How Much DNA Is Enough?

  • 500 ng of intact DNA is sufficient for a good analysis of VNTRs
  • 0.2 ng would be the minimum required for PCR analysis
  • For PCR the DNA from 50 cells is needed

PCR Analysis

  • For samples which contain insufficient, or damaged DNA, the polymerase chain reaction can be used to obtain a DNA profile
  • A specific sequence of bases in the chromosome that occurs only once is amplified as much as a million fold
  • This sequence is then identified by an indicator probe

Disadvantage of PCR

  • The sequences used for PCR do not come in as many different alleles as the VNTRs, so the probability of a random match, even after using multiple probes is higher than for VNTR profiling

Advantages of PCR

  • Samples that contain very small amounts of DNA (not capable of VNTR analysis) can be amplified
  • For example, saliva traces on cigarette butts, and single hairs containing root cells
  • Samples in which the DNA is partially degraded can be successfully analyzed
  • The method is rapid and can give results within 24 hours
  • Several different DNA sequences can be selectively amplified simultaneously
  • Sample can be reserved for duplicate testing since so little is used
  • PCR can be used to detect any kind of sequence variation in the human chromosomes, and can lead to sequence analysis

The PCR Method

  • DNA strands are dissociated at elevated temperature, eg. 94oC
  • The dissociated strands serve as templates for DNA synthesis
  • Specific primers bind to their complementary target sequences on the dissociated DNA strands
  • The primers are extended by the action of a heat resistant DNA polymerase, called Taq polymerase (from Thermus aquaticus)
  • The newly synthesized DNA is again strand-separated by heating, and the new strands now serve as templates for further rounds of synthesis
  • In other words, the first round of synthesis doubled the number of templates
  • Each subsequent round again doubles the number of available templates
  • Thus, the DNA sequence of interest is amplified in an exponential fashion
  • In practical application, between 30 and 35 cycles of amplification can be carried out yielding 107 - 109 copies of the target sequence
  • An automated computer-controlled thermocycler allows the steps to be carried out at fairly high temperatures, where the specificity of the reaction is maximized

The PCR Thermocycler

  • A thermocycler is a computer-controlled instrument which allows the primer annealing (50-65oC) and extension (70-75oC) steps to be carried out at fairly high temperatures
  • This maximizes the specificity of the template-primer hybridization
  • And optimizes the rate of primer extension

Cetus Corporation PCR Kit

  • Identifies different alleles of a protein coded for by a gene on chromosome 6
  • This protein is an HLA class II antigen
  • It is one of the antigens of the major histocompatibility complex (MHC)
  • The gene is called the DQA locus
  • 8 different alleles can be identified

Cetus Corporation DQA PCR Kit

  • The crime scene DNA is amplified using primers specific to this gene
  • Probes, specific to each allele, are immobilized on a nylon membrane
  • The PCR product is added, and the probes to which it binds are identified
  • The PCR product will bind to 2 different probes for heterozygous individuals
  • DNA from the suspect(s), the victim, and internal positive and negative controls are also run

Determining a Match

  • Calculate the probability of an individual having a particular combination of alleles in a population
  • Look up the frequency of each allele in an appropriate reference population
  • For O. J. Simpson - the African-American population

Apply the Product Rule

Multiply those frequencies together to get the probability that the observed combination of alleles could occur in the population

For Example

  • If each of five alleles was found to have a frequency in the reference population of 10%
  • Then the probability of that combination of alleles occurring in the population is: 0.1 x 0.1 x 0.1 x 0.1 x 0.1 = 10-5< /li >< /li >< /li >< /li >< /li >< /li >

The Product Rule

  • 10-5 is usually stated as: in a population of 100,000 individuals, one would expect to find this combination of alleles in one individual

Assumptions of the Product Rule

  • Allele frequencies for the genes tested are not changing
  • The different alleles being tested are inherited independently of each other - linkage equilibrium (i.e. they are on separate chromosomes)

DNA Profile Match

  • With the product rule, it is common to obtain for a five allele match a probability of 1 in 10 million