DNA STR Amplification

What is PCR?

PCR (Polymerase Chain Reaction) is an in vitro technique that uses an enzyme to make copies of a specific DNA sequence with primers determining the area of DNA that is replicated.

What are the Advantages and Disadvantages of PCR?

Advantages: Can use small and degraded samples, fast and simple. Disadvantages: Sensitive to contamination, affected by inhibitors, doesn’t give expected yields.

What are Length Polymorphisms?

Length polymorphisms are variations in the length of a DNA sequence among individuals that can be caused by differences in the number of repetitive DNA sequences.

What are STRs? How are STR Loci chosen?

STRs (Short Tandem Repeats) are short DNA sequences (2-6 bases long) that are repeated consecutively, with the number of repeats varying among individuals. Loci need to be highly polymorphic, have small fragment size, and must be independent of all other loci.

What are the Advantages of STRs?

Easy amplification, abundant in genome, highly variable, small size range, can use multiplexing and degraded DNA.

What’s the STR Nomenclature?

Name: (di,tri,tetra,penta,hexa)nucleotide. Repeat Unit: 1-6. Number of repeat units: N/Alleles (#complete repeats.#additional bases before complete unit), Base pairs (N x #bases): n. Microvariant: Incomplete repeat units.

What’s the Nomenclature for Locus, and Loci?

Locus: DaSbbb where D (DNA), a (chromosome), S (Single copy locus), bbb (number of the locus according to discovery order). Loci: assigned name w/ gene.

What Occurs in PCR Denaturation?

DNA strands separate at 90-94C, using high temperature to disrupt hydrogen bonding. GC pairs have 3H bonds (needs higher temp) and AT pairs have 2H bonds.

What Occurs in PCR Annealing?

Primers bind to their complementary DNA sequences at 59-60C.

What Occurs in PCR Extension?

DNA polymerase binds nucleotides (dNTP) to the primers, creating a new strand at 72C.

What are the components in PCR?

DNA template (1ng of evidence sample), forward and reverse primers (defines target region and uses free OH to tell polymerase where to begin), polymerase (enzyme that builds the DNA strand), Reaction mix (dNTPs, MgCl2, Buffer, BSA).

What occurs in the Final Hold/Soak?

After PCR Extension, samples are hold at 4-10C to stop the reaction and keep it stable.

What Occurs in PCR Hot Start?

AmpliTaq Gold (inactive polymerase/enzyme) is heated to cause the chemical moiety that blocks the active site in the enzyme to fall off and active it. This is before denaturation.

What Occurs in Final Extension?

Taq polymerase gets extended time to add an A to all PCR products to ensure consistent fragment lengths. Without final extension, Taq randomly adds A to some products and causes inconsistent fragment lengths.

What is the PCR cycling order and how many cycles do they go through?

Hot start (1 cycle), Denature/Anneal/Extend (25-25 cycles), Final extension (1 cycle), Final hold (1 cycle).

What does the positive and negative control contain? What’s their purpose?

Positive control: known DNA profile + checks reaction components and thermal cycler is working properly. Negative control: PCR mix and water + checks PCR is free from contamination.

What is Multiplex PCR?

Multiplex PCR simultaneously amplifies multiple STR loci in a single reaction using carefully designed primer sets with compatible melting/annealing temperatures and different fluorescent dyes, allowing loci with overlapping fragment sizes to be distinguished.

What are different ways used to detect loci in the same size range?

1: Using different dyes, 2: Change the size of products via primer redesign (creates large or smaller PCR products that don’t overlap) and mobility modifier (A non-nucleotide linker added to a primer that slows DNA movement during separation, making fragments appear larger).

What is the GlobalFiler kit?

A multiplex PCR kit that simultaneously amplifies 22 STR loci plus sex determination markers (Amelogenin, DYS391, and Y-InDel) using fluorescently labeled primers.

Why are GlobalFiler PCR products fluorescent?

The PCR primers contain fluorescent dyes that become incorporated into newly synthesized DNA fragments, allowing detection during analysis.