Culture Collections

 

Interpretation of cell line STR profiles - instrumental artefacts

Stutter

Stutter is the most common instrumental artefact and is caused by DNA slippage during amplification. Stutter occurs in between 6-10% of amplification products1.

If there is slippage in the strand which is being copied, then some of the amplified strands are 4bp shorter than the PCR product should be.

STR1

 

This can be seen on the electropherogram as stutter peak.

STR2

 

Note that the designation of the stutter product is based on the relative peak heights of the stutter product to the main peak(s) and not on the relative fluorescent units. 

In the electropherograms below, the stutter product for loci vWA and D12S391 are about 800 RFU but they are still designated as stutter because they are less than 10% of the main peaks.

STR3

STR4

 

Less frequently, DNA slippage occurs in the copying strand, a 4bp repeat is copied twice in the same strand resulting in a minor peak which is 4bp longer than the main peak(s).

STR5

The copying strand slips back 4bp while copyingresulting in one repeat being copied twice. A small proportion of amplified products are 4bp longer than the main product.

STR6

 

Incomplete adenylation

Taq polymerase always ends its amplification run with the addition of an adenine residue. However, if there is too much DNA template then this leads to some of the amplicons not ending with the terminal adenine residue which is therefore 1bp shorter than the main amplicon. This produces a characteristic ‘split peak’ on the electropherogram.

 STR7

 

Dye blobs

Dye blobs are peaks caused by the dissociation of the fluorescent tag from the primers as they break down. They are usually wider than STR peaks and can occur anywhere in the analysis and are therefore not associated with an allelic ‘BIN’.

 

Example of bleed through

The allele peak at position 10 in loci D8S1179 is above 7000 RFUs and has resulted in an off ladder (OL) peak in a different channel for loci D7S820.

 

Bleed through image 1

 Bleed through image 2

Reference

1.Leclair, B. et al., 2004. Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples. Journal of forensic sciences, 49(5), pp.968–80.

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