(protocol version 0.2)
We will do simulated experiments to test the DNA of six individuals: a mother, her three children, and two different men who may be the fathers of the children. DNA samples will be used as templates in Polymerase Chain Reaction (PCR) experiments to determine which variants each child has at certain genetic positions. A child inherits one variant from each parent, and we can usually match one up to a variant in the mother. This means the other one had to come from the father. If neither one of a possible father's genetic variants match, then he is not the father. If we examine enough genetic positions, it is very likely that we will be able to rule out any man who is not actually the father of a particular child.
When you have finished the experiment, you can look at a family tree of the individuals to confirm their relationships, and check your answers.
The place on a chromosome where a gene or other sequence of interest is located is called a locus (plural loci - from the same root as the word "location"). A particular sequence variant ("flavor") at a particular locus is called an allele. Loci that have many alleles are said to be "polymorphic", which means these loci have "many forms".
PCR ( the "Polymerase Chain Reaction") is a technique to replicate or amplify targeted segments of DNA. We will use it to amplify specific loci, then examine the amplified product to determine which alleles are present.
Electrophoresis is a method of separating molecules by size (in this case DNA) in a gel matrix in an electric field. Smaller fragments migrate the fastest through the gel, while larger fragments migrate more slowly.
A marker lane containing bands of known sizes are used to measure the sizes of sample bands. Gels have a limited range of sizes they can resolve. Molecules that are too small for the gel to resolve run as a "small" group, and those that are too big run in the "large" group. In real gels, the resolution range can be controlled by the concentration of gel material (usually either agarose or polyacrylamide) used to make the gel. In our virtual experiments, the gel resolution range will be automatically adjusted to match the range of the marker bands (actually 10% smaller than the smallest marker band to 10% larger than the largest marker band).
STRs (Short Tandem Repeats) are sequences of DNA composed of multiple copies of a particular base sequence. "Short" repeats are one to six bases in length. "Tandem" means they are arranged head-to-tail (as opposed to "inverted repeats", or palindromes). For example, two tandem repeats of the short tetranucleotide sequence "CTAG" would be "CTAGCTAG". An STR locus is a place in the DNA where an STR occurs. For our purposes, we will focus on STR loci in which alleles vary in the number of repeats.
A big advantage of STR loci is that we can tell alleles apart simply by comparing their sizes on an electrophoresis gel. (In contrast, for alleles that are the same size but have different sequences, you might need to do a more complicated experiment, like hybridization or DNA sequencing, to tell them apart.)
There are thousands of STR loci in the human genome. The FBI has chosen a standard set of 13 STR loci to use for identifying DNA from crime scenes and from known criminals. Because they use the same set of loci for each DNA sample, they can store the results in a database called "CODIS" (Combined DNA Index System). Each locus has one or more standard sets of PCR primers used to amplify it. The alleles for each CODIS locus are well studied, and are documented at http://www.cstl.nist.gov/biotech/strbase/fbicore.htm
None of the CODIS loci are genetically linked to one another. They sort independently, which means that inheriting one allele at one locus does correlate with inheriting another allele at a different locus.
D1S80 is another repeat locus, but since its repeats are so long (16 bases), it is technically not an "STR", but a "VNTR" (Variable Number of Tandem Repeats) locus. This locus is sometimes used in teaching labs, because the alleles are spaced widely enough to be resolved on a relatively low-resolution gel. It is not commonly used in actual forensic work, though, because the bands are too big to be resolved conveniently on automated equipment set up for using high resolution gel materials to determine STR alleles.
Amelogenin is a locus on the X and Y chromosomes. The version on the Y chromosome is shorter than the one on the X chromosome. Unlike the polymorphic STR loci, we only look at two versions of amelogenin (the X and the Y alleles). Because the PCR product from the X chromosome is larger than the product from the Y chromosome, males (XY) will have two bands and females (XX) will have only one (the larger band).
You can find the PCR simulator web page for this exercise by following the link at http://www.cybertory.org/exercises/#paternity . The page has three sections. The first section, "Marker", specifies the DNA size standards. These markers are "ladders" of evenly spaced bands. You can enter sizes (in DNA base pairs) for the smallest band in the ladder, the largest band, and for how the spacing between "rungs". These can be adjusted based on the expected sizes of PCR products.
The second section sets the "Default Reaction Parameters". PCR reaction parameters chosen in this section will be specified as defaults for all of the reactions.
The third section, "PCR Setup", allows you to select reaction conditions for each PCR reaction separately.
At the bottom of the page is the "run reactions and show gel" button. To see an animated gel, the Adobe SVG plugin must be installed. Go to the Adobe website (www.adobe.com) to install the plugin. You can see static images of your gels without installing the plugin by selecting "JPEG" from the output format menu.
The six simulated people we will test in this exercise are listed in Table 1. Samples in the PCR simulator are labeled by "patient number", so you can use this table to tell who the people are.
Table 1: Individuals for Paternity Testing| Person | ID |
| Mother | (Patient #04) |
| Possible Father A | (Patient #05) |
| Possible Father B | (Patient #06) |
| Child A | (Patient #07) |
| Child B | (Patient #08) |
| Child C | (Patient #09) |
Our first PCR experiment will determine the sex of each individual using the Amelogenin primers. We need to test the six people from Table 1, but we have eight reactions available, so we might as well test some extra, unrelated people as controls. We will use the same primers and conditions in each reaction, but different templates.
To get a definitive paternity identification, PCR results for the FBI CODIS STR loci need to be determined. The FBI CODIS PCR primers are all available on the PCR webpage as options in the Primer A and Primer B pulldown menu.
To confirm the identity of the father of the children, examine the family tree. The mother's name is "Sharron Goddard". You can click the links to her children, who will have links to their fathers. The "patient IDs" in the family tree correspond to the patient IDs for the PCR templates. Did you pick the right father for each child? How might you explain your results if they do not agree with the family tree?