Biology 442 - Human Genetics

Materials Used

(Cells with nuclei):
Blood cells (lymphocytes
Semen, tissue, urine
Buccal swabs, saliva
Pre and postnatal: CVS and amnio, cord blood
Hair follicle cells

Techniques Used

DNA extraction
Restriction enzme digestion
Southern Blots
PCR
Liquid chromatography

The ABO and HLA genes

The ABO blood type locus was used in identity testing early on. However, since there are only three alleles (A,B, and O), it had very limited ability to identify or exclude persons. Exclusion of relatedness is possible in some cases, for example when the individual in question is type AB and the child or forensic sample is O. Also before DNA testing was possible, cell surface proteins (antigens) were used. These are coded for by the highly polymorphic loci in the HLA (human leukocyte antigens) region of chromosome 6. These loci have 10 - 50 alleles per locus but the cell surface proteins are technically difficult to test forensic they require the use of specific antibodies.

DNA Polymorphisms

The current techniques employ DNA polymorphisms at first using RFLPs (Restriction Fragment Length Polymorphisms), then VNTRs (Variable Number of Tandem Repeals) and STRs (Short Tandem Repeats). DNA fingerprinting requires highly polymorphic markers for which most individuals are heterozygous. These markers are inherited in a simple Mendelian, codominant fashion. A polymorphic locus is one in which there exists a significant variety of alleles in the human population at that locus. The polymorphic markers used in DNA fingerprinting must be "unlinked" independently inherited loci. The loci used are in non-coding regions of the DNA and since non-coding DNA is significantly more abundant than coding DNA there are many such loci to use.

Southern Blots

Detection of these polymorphic loci requires digestion of the DNA by restriction enzymes followed by electrophoresis and Southern blotting of the DNA onto nitrocellulose paper, denaturing the double stranded DNA to single stranded, "blocking" with control DNA, and finally, the application of a labeled probe (e.g., radioactivity or chemoluminescense) to the blot, incubation and washing. Probes are specific, known DNA sequences. Multi locus probes can be the VNTR (or other repeat) itself, in which case there are many bands which look like a "bar code" in a supermarket. A single locus probe is a DNA sequence known to be nearby the tandem repeat and unique to that chromosome and region. Southern blots require a rather large quantity of DNA which is in good condition (not contaminated, not digested or degraded, etc.).

Here's an example of one kind of DNA testing procedure using Southern blots:

1. Evidence: DNA is extracted from blood, hair, semen or other body tissue.

2. Fragmentation: Using a restriction enzyme, the DNA is cut into fragments. The fragments vary in length depending on an individual's genetic code.

3. Separation: The fragments are placed in a tray containing special gel. An electrical current pulls the fragments along the gel. Heavier fragments move a short distance; lighter fragments go farther. As a result, the fragments may be separated according to size.

4. X-ray: The separated DNA is transfered to a nylon membrane and radioactive or chemoluminescent DNA probes are added. These genetic "probes" search out and lock onto complementary regions of the DNA. An X-ray or pictures is made of the matching fragment patterns on the membrane.

5. Autorad: The X-ray or picture, called an autoradiograph or autorad, is developed, producing a pattern of bands that look like a bar code. This DNA "fingerprint" is compared with others for matches.

PCR

PCR has become the method of choice when the proper equipment is available. PCR requires less DNA but theoretically a single cell worth of DNA can suffice and it can even be partially degraded. Amplification of the desired DNA sequence requires knowledge of the DNA sequences of the regions flanking the repeat region in order to construct the appropriate PCR primers. The Taq DNA polymerase (taken from Thermus aquaticus) can only add dNTPs to the primers. About 25 to 40 cycles provide sufficient material. Amplification can occur only over a few hundred base pairs. Nested PCR is used for degraded samples. Multiplex PCR and differently color tagged primers are used when looking simultaneously at more than one locus. The PCR products are electrophoresis in gels. PCR can also be used on coding regions if the allele sequence differences are known (DQ alpha with 6 alleles; ABO, HLA). In these, the exon of interest is amplified.

Likelihood of Identity Determination

The theory of identification is simple and straightforward: a sample from an individual is compared to close biological relatives or a crime scene sample. In the absence of mutation, the closest relationships are parent-child followed by full sibs who should, on average, share half of all alleles. However they can be expected to lack shared alleles in up to 25% of loci (i.e., 1/2 x 1/2 = 1/4). The Likelihood of relationship or Likelihood Ratio (LR) is calculated to estimate the significance of the biological relationship. LR = A. probability of the DNA typing results if the individual is the person, parent, child or sib of the reference DNA sample divided by B. the probability the putative relationship is not genuine and only due to chance match between two unrelated individuals. B requires the knowledge of allele frequencies in the relevant population. The higher the degree of polymorphism at each locus tested (i.e., the more alleles that exist and the lower the frequency of each allele in the population), the less the likelihood of a "chance" match between unrelated individuals.

Catch a Criminal           Create a DNA Fingerprint          Creating A DNA Profile

Calculation of the Paternity Index

Is the man excluded? Yes, if the bands do not match; No, if they do. There are only two reasons why the DNA profiles would match. Either the person is the person we suspect or there are two different and unrelated individuals who share this genetic pattern. In the latter case we need to determine how often that DNA profile might occur in the general population. Population data bases are converted to tables indicating what percent of the population has a particular band. After the individual frequencies for each gene pair is calculated, they are multiplied together since they are independent events (unlinked loci). Different loci are examined until the person is excluded or until you get to the probability you have determined you need to be certain. Courts use 99% but you can use 99.9 or 99.8% as you decide. 99.9% means 999/1000 or 1000 X more likely to be the person than not. 99% means 100X more like to be the person than not.

Y Chromosome DNA and Mitochondrial DNA

The use of the DNA of the Y chromosome and the mitochondria offers unique advantages. The Y chromosome is passed exclusive through the males and the mitochondria are (with rare exception) inherited only from the mother since male derived mitochondria are destroyed in the fertilized egg. The Y chromosome and the mitochondrial DNA also have no ability to recombine and thus are simpler to study.

Genes unique to the Y chromosome are useful in establishing the sex of a dead individual. In addition, DNA polymorphisms of the Y are useful in tracing paternity. The Y chromosome is small, with poor gene content. About half of the q arm is heterochromatic and there are no genes there. Thirty percent of the chromosome is euchromatic. Except for the Yp arm, it does not recombine and is passaged only through males. Therefore, it is useful in human evolution population studies and DNA identity testing.

Polymorphisms on the Y chromosome were used to identify Thomas Jefferson as the father of his slave Sally Henderson's children by comparing the Y chromosome of male descendents of the male offspring of his brother and the Y chromosomes of Sally's male children's male descendents. Thomas Jefferson had no male children by his legal wife so it was necessary to use his brother's descendents to determine relatedness of Sally Henderson's descendents to him. This strategy was based on the fact that Thomas Jefferson and his brother shared the same Y chromosome they inherited from their father.

As mentioned in an earlier lecture, comparison of the sequence of mt DNA from the skeleton believed to Czar Nicholas, with the exhumed bones of his younger brother, and the mitochondrial DNA (mtDNA) from two living relatives showed a rare mt DNA mutation present heteroplasmically in the two brothers' skeletons and homoplasmically in the two living relatives (see lecture on non-traditional inheritance for an explanation of hetero- and homoplasmy). There are many examples of human evolution studies and forensic uses of mt DNA. Mitochondria DNA is inherited exclusively from the mother, therefore it can be used to identify persons related through a common female. MItochondrial DNA (mtDNA) comparisons are usually made from the DNA of the D-loop (D is for displacement) which, incidentally, has a triple DNA stranded structure. The D-loop is a non-coding region and contains the origin of replication site as well as the primary promoter for transcription. Mitochondrial DNA mutates more rapidly than nuclear DNA probably due to the lack of repair enzymes and the greater exposure to free radicals within the mitochondrion. It is useful for relatedness studies because cells contain 100's - 1000's of mitochondria and mtDNA is therefore easy to extract.