PCR Reagents | Stochastic Effect | STR Classification

PCR Reagents

  • Tris CI (pH 8.3)                             10-50mM
  • Mg CI2                                           1.2-2.5mM
  • KCI                                                 50mM
  • dNTPs                                            20μM(each)
  • DNA polymerase (thermostable)      5-5units
  • BSA                                                 100mg/ml
  • Primers                                          0.1-1um (each)
  • Template                                       1-10ng
  • Water (deionized)               Depends on end concentration (how much reaction is to be carried)

BSA is added because when there is a single purified protein then its stability will be less. When BSA is added stability will increase. Taq polymer rase will perform better. When there are some proteases then by adding BSA it can be protected it will keep proteases engaged so in this way the target protein/ DNA will be protected.


Stochastic Effect

For one STR, there should be 2 peaks:
As there will be STR on both chromosomes (one from father, one from mother). If homozygous then there will be one peak (this should not happen). Those STR are selected which have more chances of variation. There are some criteria to select those STR sites which have more power of discrimination.

If in all individuals, there will be the same copy number then it's of no use. If 13 STR sites do not have much power of discrimination than use more. If the copy number in genomic DNA is less than 17 than there are more chances of allele dropout.

If there is one copy of DNA then there will be 3 picograms of DNA If two copies there will be 6 program means 10 cells.

Primers are designed for the flanking region of STR sites and it should show some stability.


Stable primers should be designed. Fewer chances of mutation. Stable flanking regions. Such should not be selected which will change. Due to allele dropout, one peak will come and are may think that it's homozygous but actually it was heterozygous. Thus the recommended amount is 1-2.5ng.

STR Classification

The more the repeat size increases the less the chance of its occurrence.

  • Simple Repeats: One STR site repeating again and again. There is a range of copy numbers e.g 4-9 etc.  It is known that in what range product will come (the exact size is not known).
  • Compound Repeats: Two or more than two repeats at some site together.
  • Complex Repeats: More than 3 or 4 repeats coming together. Blocks of variable length repeat composed of several repeats.
  • Hyper Variable Complex Repeats: Not used from a forensics point of view. There will be some other sequences with repeats. Several different types of repeats along with other sequences e-g transposable elements.

Forensic Science mostly uses simple or compound repeats. In some cases, complex repeats may also be used.

Forensic Samples are challenging to work with: 
Problems
  • Degradation
  • Contamination
  • Limited/small amount of DNA to deal with.
  • Mixed samples as in the case of sexual assault.
  • Bacterial and fungal contamination is not important as there are no STRs in them
  • Primate DNA contamination should not be there if so then it should be known how to differentiate.
Editor's Recommendation:

PCR Reagents | Stochastic Effect | STR Classification PCR Reagents | Stochastic Effect | STR Classification Reviewed by AB Ultimate Guides on June 17, 2020 Rating: 5

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