STR/YSTR
Name three advantages/situations to testing YSTR over autosomal STRs.
1. In cases where there is a mixture where the amount of female DNA is much higher than the amount of male DNA. YSTRs allows you to essentially ignore the female component of the mixture and target the male DNA only. 2. In cases where demonstrating paternity of a male is important. 3. In situations where there is a complex autosomal STR mixture, it may help distinguish how many male contributors there are.
Name two disadvantages to testing YSTRs.
1. The inability to distinguish male relatives from one another since the Y chromosome is inherited in its entirety from father to son. 2. Because it is a haplotype and all of the alleles are linked since they are located on the same chromosome, the statistics involved aren't going to be as discriminatory as the statistics involved with STRs.
Are there core YSTR loci? How many?
17, need 15 or more Y-filer loci for SDIS and no dropout at 385a/b
How long are the core repeat units for STRs?
2-7 bp in length, but we typically stick with tetranucleotide repeats.
Explain what amelogenin tests for and what is the premise for its usefulness?
Amelogenin is a gene that codes for proteins found in tooth enamel. Within intron 1 of this gene, the X homologue has a 6bp deletion. The primers flank this region and will result in either a 106 or 112bp amplicon from the X or Y chromosome, respectively. This difference is useful in helping determine the gender of the subject. This can be useful to help distinguish between victim and suspect or gender identification.
What is a microvariant allele? Give an example and explain the allele nomenclature.
Microvariant allele = Alleles that contain incomplete repeat units. An example of this would be the allele 9.3 at the TH01 locus, which contains nine tetranucleotide repeats and one incomplete repeat unit consisting of 3 nucleotides, hence the ".3" (the number after the decimal is the number of nucleotides in the incomplete repeat unit).
How many alleles are typically observed at YSTR loci? Name three exceptions.
One 1. Mixture of YSTR DNA types 2. Duplicated Marker where the duplicated regions are different lengths (DYS385a/b) 3. Duplication at particular loci (see J Forensic Sci, July 2005, Vol. 50, No. 4 - in module 12 folder) 4. DYS389I/ DYS389II - Honorable mention since it is technically two locations in the profile
What does STR stand for?
STR = Short Tandem Repeats
Where are the STRs that we use in forensic testing typically located in the DNA genome? Why are they located there?
STRs are usually located in non-coding regions of the genome and may be found surrounding the chromosomal centromere. These repeat sequences are typically found between genes, in non-coding regions, because they can vary in size from person to person without impacting the health of the individual. This allows for the high level of variability within these regions.
What are "core" STR loci? How many are there?
The Core STR loci are a common set of standardized loci. This is important to establish for consistency across a wide number of jurisdictions. If different agencies were looking at different loci, it would be impossible to compare data within a database. There are 13 core loci that were chosen to be the basis of the CODIS national database. Now there are 20.
How many alleles are typically represented in the DYS385 locus? Why?
DYS385 typically has 2 alleles because it is present in 2 locations on the Y chromosome. The duplicated regions are located ~40,775bp apart facing away from each other. A single set of primers binds both regions, giving 2 alleles.
Characterize what is happening at the DYS389 loci.
DYS389I is a subset of DYS389II. The way that this happens is that there are 2 binding sites for the forward primer and only one binding site for the reverse primer, so you end up with 2 fragments that are copied.
YSTR markers are independently inherited. True or False?
False. All Y alleles are on the same chromosome and are linked.
Can male DNA be masked by female DNA (explain your answer in terms of both autosomal STR and YSTR)?
In the case of STRs, yes, the male DNA can be masked by the female DNA when the amount of female DNA present is much more than the male DNA. The female DNA will amplify in a larger quantity making the relatively small amount of male DNA undetectable. For Y-STRs, no. The male DNA should be the only DNA amplifying and will, therefore, be the only DNA detected effectively removing the female DNA from the equation.
Why do the preferred STRs used for forensic DNA analysis contain tetranucleotide repeats as opposed to di- or trinucleotide?
The reason we use tetranucleotide repeats the most is because the amount of stutter observed is less than with a di- or trinucleotide repeat (data with less stutter is easier to interpret). Also tetra's give a 4 bp spread between alleles which makes it easier to resolve heterozygotes with a size-based separation method. Penta and hexa repeats are less common.
Why is it important to be aware of mutations in YSTRs?
There are occasionally mutations that happen in YSTRs, so it is important to be aware of this because a difference may occur between a father and a son (or other paternally related male). This is typically seen with a single repeat addition or deletion at one or two loci. It is important to have mutation rates for the YSTR loci in order to interpret the results and report stats.
Are the STR loci currently used in our lab human specific?
They are specific to humans but will produce results for primates. Results from a primate are distinguishable from a human by their number of off ladder peaks and the irregular, imbalanced nature of amplification across the multiplex.
Do STRs vary by sequence or length?
They vary in length
What type of length polymorphisms were tested by using RFLP
VNTR (variable number tandem repeats)
What does VNTR stand for?
VNTR = Variable Number of Tandem Repeats
Is YSTR testing considered haplotype testing? If so, why?
Yes, it is considered haplotype testing because there will be only a single allele at most loci. This is because males only have 1 Y chromosome (females don't have one at all). Alleles are inherited from a single parent together.
Do paternal relatives have the same YSTR profile? Please explain.
Yes. A father will pass his entire, intact, Y chromosome to his male children (barring the occurrence of a mutation). So, the male children will have the exact same YSTR profile as each other and their father.
Are known female standards amplified with the Y23 kit? Why or why not?
Yes. The reason for this is that we sometimes get YSTR results with female DNA. It is important to know this so that, if there are results for the male loci within the STR kit, we will know that they are not necessarily from a male within the sample.
What are the main advantages of using PCR over RFLP for forensic analysis?
a. For PCR only small amounts of template DNA is needed. b. Degraded DNA can be used as template. c. Large numbers of copies of specific, relatively small (~100 to 400 bp) DNA sequences can be amplified simultaneously using PCR. d. Contaminant DNA (bacteria or fungal) will not be amplified because of human specific primers. e. Commercial kits make for quick, easy setup. f. PCR can be easily automated and it's a much faster method than RFLP.
List the different classifications used to describe the complexity of the STR core repeat sequences. Give an example for each type.
• Simple Repeats contain units of identical length and sequence (TPOX, CSF1PO, D5S818, D13S317, D16S539) GATA-GATA-GATA-GATA-GATA-GATA • Simple, Non-Consensus Repeats (D18S51, D7S820, TH01) GATA-GAT-GATA • Compound Repeats comprise two or more adjacent simple repeats (vWA, FGA, D3S1358, D8S1179) GATA-GATT-GATA-GATT • Complex Repeats contain repeat blocks of variable unit sequence/length units (D21S11) GATA-GACA-CA-CATA • Complex Hypervariable Repeats contain numerous non-consensus alleles that differ in both size and sequence (SE33) [AAAG]3, [AAAGG]1 and [AAAAA]1 combined with a variable number of [AG]n and [AAAG]n
