Isolation And Types Of Str Markers

In order to perform analysis on STR markers, the invariant flanking regions surrounding the repeats must be determined. Once the flanking sequences are known then PCR primers can be designed and the repeat region amplified for analysis. New STR markers are usually identified in one of two ways: (1) searching DNA sequence databases such as GenBank for regions with more than six or so contiguous repeat units (Weber and May 1989, Collins et al. 2003, Subramanian et al. 2003); or (2) performing molecular biology isolation methods (Edwards et al. 1991, Chambers and MacAvoy 2000).

STR repeat sequences are named by the length of the repeat unit. Dinucleotide repeats have two nucleotides repeated next to each other over and over again. Trinucleotides have three nucleotides in the repeat unit, tetranucleotides have four, pentanucleotides have five, and hexanucleotides have six repeat units in the core repeat. Theoretically, there are 4, 16, 64, 256, 1024, 4096 possible motifs for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively (Jin et al. 1994). However, because microsatellites are tandemly repeated, some motifs are actually equivalent to others (D.N.A. Box 5.1). For reasons that will be discussed below, tetranucleotide repeats have become the most popular STR markers for human identification.

STR sequences not only vary in the length of the repeat unit and the number of repeats but also in the rigor with which they conform to an incremental repeat pattern. STRs are often divided into several categories based on the repeat pattern. Simple repeats contain units of identical length and sequence, compound repeats comprise two or more adjacent simple repeats, and complex repeats may contain several repeat blocks of variable unit length as well as variable intervening sequences (Urquhart et al. 1994). Complex hypervariable repeats also exist with numerous non-consensus alleles that differ in both size and sequence and are therefore challenging to genotype reproducibly (Urquhart et al. 1993, Gill et al. 1994). This last category of STR markers is not as commonly used in forensic DNA typing due to difficulties with allele nomenclature and measurement variability between laboratories, although two commercial kits now include the complex hypervariable STR locus SE33, sometimes called ACTBP2 (Urquhart et al. 1993, Promega Corporation 2002, Applied Biosystems 2002).

Not all alleles for a STR locus contain complete repeat units. Even simple repeats can contain non-consensus alleles that fall in between alleles with full repeat units. Microvariants are alleles that contain incomplete repeat units. Perhaps the most common example of a microvariant is the allele 9.3 at the TH01 locus, which contains nine tetranucleotide repeats and one incomplete

D.N.A. Box 5.1 List of possible microsatellite motifs

Theoretically, there are 4, 16, 64, 256, 1024, 4096 possible motifs for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. However, because microsatellites are tandemly repeated, some motifs are actually equivalent to others. Two rules can be used to identify whether motif A is equivalent to motif B. Motif A is considered equivalent to motif B when (1) motif A is inversely complementary to motif B, or (2) motif A is different from motif B or the inversely complementary sequence of motif B by frameshift. For example, (GAAA)n is equivalent to (AGAA)n or (AAGA)n, to (AAAG)n or (TTTC)n, to (TTCT)n or (TCTT)n, or to (CTTT)n. In other words, the eight motifs are equivalent. Note that (AGAG)n is considered a dinucleotide repeat instead of a tetranucleotide motif (Jin et al. 1994).

Because of this equivalence in repeat motif structure there are only 2, 4, 10, 33, 102, and 350 possible motifs for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively (see below).

Mononucleotide repeats (2): A C

Dinucleotide repeats (4): AC AG AT CG

Trinucleotide repeats (10): AAC AAG AAT ACC

ACG ACT AGC AGG ATC CCG

Tetranucleotide repeats (33): AAAC AAAG AAAT AACC AATG AATT ACAG ACAT ACTC ACTG AGAT^ AGCC ATGC CCCG CCGG^

AACG ACCC AGCG

AACT ACCG AGCT

AAGC ACCT AGGC

AAGG ACGC AGGG

AAGT AATC ACGG ACGT ATCC ATCG

AGAT or GATA motif is the most common for STR loci used by forensic scientists

Penta- (102) and hexanucleotide (350) repeats are not shown due to the sheer number of motifs possible.

Source:

Jin, L., Zhong, Y. and Chakraborty, R. (1994) The exact numbers of possible microsatellite motifs [letter]. American Journal of Human Genetics, 55, 582-583.

repeat of three nucleotides because the seventh repeat is missing a single adenine out of the normal AATG repeat unit (Puers et al. 1993).

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