Protocol:Checking for maternal DNA contamination by STR analysis

Checking for maternal DNA contamination by Short tandem repeat (STR) analysis 

principle

Routine examination of short tandem repeats (STR’s) or Variable Number of Tandem Repeats (VNTR’s) in fetal and parental DNA samples by PCR analysis is essential to rule out maternal contamination.

The tandem repeated “minisatellite” regions of DNA are an ideal method to identify maternal contamination. These highly polymorphic DNA regions show alleleic variation in the number of repeated units. Because of the large number of different alleles these repetitive DNA areas provide informative genetic markers. Since these variations are inherited according to Mendelian genetics, they can be used to test for maternal contamination and indeed will also identify non-paternity

It is recommended to check for maternal DNA contamination in every case of prenatal diagnosis especially when the fetal genotype is the same as mother's genotype. The choice of polymorphic markers available is wide, including the short tandem repeat (STR) markers such as D21S11, D21S1414, D18S535 (1) or Variable Number Tandem Repeat (VNTR) markers such as ApoB, IgJH and Has-ras (2). Using the quantative fluorecesent (QF—PCR) described below trisomies may also be detected particularly tri-allelic patterns (3)

With the use of unique fluorescent oligonucleotide primers flanking the repeat sequence, the polymorphic regions on the chromosomes can be amplified using Gap-PCR. The PCR products are separated and analysed using an automated laser DNA analyser (ABI Prism 310 or 3100), and the appropriate genescan software.

Reagents

 * 1) PCR mix (8 ml)

1 ml 10 x cetus buffer

5.4 ml sterile distilled H20

1.6 ml 1.25 mM NTPs

8 ml spermidine 1 M

Store frozen in 1 ml aliquots

Final concentration of PCR reaction: 50 mM KCl, 10 mM Tris, 1.5 mM MgCl2,0.01% gelatin, 200 M each dNTP


 * 1) 10 x Cetus buffer

1.25 ml 2 M KC1 (stock solution)

0.5 ml 1 M Tris pH 8.3 (stock solution)

75 ml 1 M MgC12 (stock solution)

5 mg gelatin (300 bloom)

3.2 ml sterile distilled water

Place at 37 oC to dissolve gelatin

Store frozen


 * 1) Stock solutions

2 M KC114.91 g/100 ml

1 M TRIS12.11 g/100 ml adjust pH to 8.3 with concentrated HCl

1 M MgC12 6H2020.33 g/100 ml

Store at 4 oC


 * 1) dNTPs 1.25 mM - 100 mM conc)

60 ml of each dNTPs (dATP, dCTP, dGTP, dTTP)

4.74 ml sterile distilled water

Store frozen


 * 1) Spermidine

Free base obtainable from Sigma (S:2626)

Prepare 1 M solution in distilled water (MW:145.2)

Store frozen

Primers
All primers for are diluted to O.D. of 1, ie approximately 10 pg/ml, and stored frozen. Table 7.1 shows the primers used in multiplex QF-PCR. Seven STR markers are described here. In each case the forward primer from each set is labelled with a fluorescent dye (3,4). The primers are optimised to ensure comparable amounts of PCR products. In addition markers on the Amelogenin gene (AMDXY) can be used. The use of these markers enables sexing of the fetus. The Y chromosome produces a fragment of 252 bp and the X chromosome a fragment of 432 bp (5,6)

Fluorescent PCR (QF-PCR) Method
protocol


 * 1) To a 0.5 ml Eppendorf tube add:

20 l PCR mix

Forward labeled primer (For concentrations see table 7.1)

Reverse primer (see table 7. 1)

0.1 l- 0.5units taq polymerease (Perkin Elmer)

1 l 1/10 dilution of DNA

1 drop of mineral oil

Make up to 25l with deionised distilled water


 * 1) Place in thermal cycler programmed as follows:

93 C for 48 seconds

60 C for 48 seconds

72 C for 1 minute

For 25 cycles

72 C for 3 minutes

Refrigerate


 * 1) Add 1 l amplified product to 12 l formamide and 0.5 l size standard (Genescan 500 TAMRA or Rox size standard if using the ABI 3100), in 0.5 ml sample tube that do not have lids (available from ABI), making sure that there are no bubbles. Place grey septa (from ABI) on each tube
 * 2) Place at 92 C for 2 minutes. The PCR products are run through a capillary (15 kVolts, 24 min at 60 oC). The specific PCR products are sized and the amount of each PCR product evaluated by the extent of fluorescent activity, equal to the area of the fluorescent peak generated.

Interpretation of results
The peak height should ideally be between 100 and 1000. For peak heights <100, the product should be amplified for a further 5 cycles and re-analysed.

Table 7.2 shows how to analyse the fluorescent markers on the ABI genescan and the appropriate size standard to use.

Muliplex PCR method
Primers can also be multiplexed; the following method is for D21S11, D13S631, D18S535 and AMDXY primers

protocol


 * 1) To a 0.5 ml Eppendorf tube add:

2.5 l 10x PCR (supplied with Amplitaq)

1.5 l 25 mM MgCl2 (supplied with Amplitaq)

4 l 1.25 mM dNTPs

Appropriate volume of D21S11 F and R, D13S631 F and R, D18S535 F and R, AMDXY F and R. (See table 7.1)

0.2 l -1 unit taq polymerase (Perkin Elmer)

1 l 1/5 dilution DNA (originally approximately 0.5mg/μl)

1 drop mineral oil

Make up to 25 l with deionised distilled water


 * 1) Amplify as above but with an extension time of 30 minutes at 60 C after 25 cycles. Analyse on ABI 310 or 3100 as above, except analyse the red size standard and the other relevant colors relating to the STR markers used.
 * 2) If any of the markers do not work very well, repeat using the primers for that STR separately.

Table 7.1. Primers used in the multiplex QF-PCR

Table 7.2 showing the analysis of the short tandem repeat markers on the ABI Genescan