Desirable Characteristics of STR used in Forensic DNA typing

Desirable Characteristics of STR used in Forensic  DNA Typing

There are 8000 types of STR but all are not useful in forensics. Only a limited number of STRs are used for forensic DNA typing. First of all size of the STR product should be known. It is usually about  100-200bp. VNSTR product size is in the range of 400-1000bp. (Minisatellites or VNSTRs are used in RELP). Size can be more than it as well. It depends upon the region against which primers are designed in the region between primers (length of primers).

For sequencing of a PCR product whole product is not sequenced i-e sequence may not be up to complete range. For example, up to 1.25, 1.5kh but not 2kb. In the dideoxy method, there is a limitation that the larger the product the sequencing will not be possible.
As the dideoxy method depends upon addition so where there will be primers, the addition will occur next to it.

Sequencing will our next to primer. For full sequencing, primers should have to be designed against flanking regions.

The sequencing of the STR product will not be carried. On the base of product size, the copy number is seen. The system should be efficient with high resolving power to distinguish:

• Resolving power is more in capillary electrophoresis.

• The page has more resolving power and if the pore size is less & length is more then minute differences can be seen.

The method should give the result in the shortest period of time.

The single base pair difference can be resolved by Denaturing Gradient Gel  Electrophoresis (DGGE). The principle of DGGE is that a denaturing agent like urea is added and a continuous gradient of gel is formed. When DNA moves then the amount of denaturing agent increases. At a certain point, DNA will come in single-stranded form i-e gets denatured. In a molecule, where there is a mutation, denaturation will occur more quickly as compared to these which have no mutation.

STR may be di to hexanucleotide repeats but tetra are more in use in forensics. Hexa and hepta have fewer frequencies. The highest will be di and tri but in use are tetrad they perform well. Penta & Hexa are less common.

Stutter Product Formation:
There is a problem with stutter product formation. This problem is 15% in tetranucleotide repeats and 30% in di and tri repeats. Two peaks appear instead of 1 one is of smaller size. Copy number will change sometimes it may come sometimes not (mostly less number) so an extra peak forms. This is a stutter product and causes difficulty in the interpretation of results.  Thus, such repeat is taken in which the chances of stutter products will be less. Therefore, tetra is used. Penta & Hexa cannot be used because they are already less in number.

Narrow Allele Size Range:
Another characteristic of tetranucleotide is that it has a narrow allele size range. Tri & di have wide copy number change. Such is required whose copy number will be less. Thus, through whole population statistics, it is seen that STR will have less range. Hence, tetra is taken as there is a little difference in copy number not in hundreds.

1. Preferential amplification of smaller alleles.

2. Narrow allele size range.

3. Smaller product size enables us to work with degraded DNA.

4. Reduced Stutter Product Formation.

5. The power of discrimination heterozygosity should be >70%.
If repeats don’t change in all individuals then it cannot be used.

6. Separate chromosomal location.
Should be on different chromosomes.not together in the same region. sequences should not be too near on the same chromosome

7. Low mutation rate.
One mutation is that is which copy number is changing. This is a desirable character. The other mutation is that in which flanking region changes. This should have a low rate because if flanking region changes then primers will not bind & there will be no amplification. Thus, this region should be stable. Such STR sites whose flanking region is changing are of no use.
Copy number should change even between siblings & parents offspring. Individuals should have their own signatures.

8. The predicted length of the allele size should be 90-500 bp.
Such STR sites whose product comes in this range.

Multiplex PCR is used in STR analysis

Within the same tube, multiple reactions are carried using multiple primers. Within the same tube, different products are obtained using a small amount of DNA. If two products have the same length then through different labeled primers a mixed product can be resolved. Separate PCR can be used as well. The band size can also tell that two sites have mixed here but which two sites this cannot be known.

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