Variable number of tandem repeat (VNTR) and short tandem repeat (STR) sequences are inherited repeating stretches of DNA from hundreds (VNTRs) to a few (STRs) nucleotides generally found in the non-coding regions of the human genome. These stretches of DNA show size heterogeneity among humans and are inherited in a co-dominant fashion. In the test, oligonucleotide primers, flanking a VNTR/STR, are extended using the PCR. Agarose or polyacrylamide gel electrophoresis is used to estimate the PCR fragment sizes, respectively VNTR and STR.


Extracted DNA (Protocol 3.6); maternal, paternal (optional) and amniotic fluid or cervical smear DNA.

Equipment and Materials

Microcentrifuge Thermal cycler Micropipettors (general use) Micropipettors (PCR use only) Filter barrier micropipette tips 0.2 mL PCR tubes 100 mM dNTP 10 x PCR buffer 25 mM MgCI,

VNTR primers (Genome Database: D1S80-PCR1.1/PCR1.2 (GDB:1 78639) 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3' 5'-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3' D1 7S5-YNZ22.1/YNZ22.2 (GDB:1 78624) 5'-CACAGTCTTTATTCTTCAGCG-3' 5'-CGAAGAGTGAAGTGCACAGG-3' ApoB-PCR6.1/PCR6.2 (GDB:177687) 5'-GGACAGTGAAACGAGGGC-3' 5'-GGCACATGAAGACACCAGAGG-3' STR primers (Maxim Biotech, Inc., San Francisco, CA): HPRT (Cat #STR-1001/STR-1002) Tyrosine hydroxylase (Cat #STR-1003/STR-1004) Androgen receptor (Cat #STR-1007/STR-1008) Intestinal Fatty Acid Binding Protein (Cat #STR-1009/STR-10010) HotStarTaq Polymerase (Qiagen Inc., Cat #203205) or Taq polymerase


1. Prepare the appropriate cocktail (see PCR Cocktail below) for the total number of samples to be analysed. Make up a volume that is -10% greater than the total volume required (include the samples, controls and a water blank).

2. Aliquot 30 pL of PCR cocktail into 0.2 pL PCR tubes.

3. Add 250 ng of DNA, vortex and flash spin tubes.

4. Amplify according to the PCR profile below.

5. Combine 10 pL of the PCR product with 2 pL of 6 x loading buffer and electrophorese using 3.0% NuSieve agarose (for VNTR, see Protocol 3.11) or 10% polyacrylamide gel (for STR, see Protocol 3.12).

6. Photograph the gel. The expected PCR products should be clearly visible. If no bands are visible, or there are unexpected bands refer to the troubleshooting checklist in Part 2 (Table 2.4).

PCR cocktail


One sample


10 x buffer

5.0 pL


25 mM MgCI,

3.0 pL


2 mM dNTPs

5.0 pL


Sense primer (2.5 nmol)

1.0 pL


Antisense primer (2.5 nmol)

1.0 pL


HotStarTaq polymerase (5.0 U/pL)

0.4 pL


Sterile ddH.O

14.6 pL



30.0 pL

94°C for 5 minutes for Taq polymerase, or 15 minutes for HotStarTaq. 94°C for 30 seconds, 60°C for 60 seconds, 72°C for 120 seconds for 35 cycles.

Procedural Notes

See Protocol 3.8 and Part 2 for precautions to help avoid contamination.

56 Molecular Protocols in Transfusion Medicine Quality Control

1. Contamination is indicated by testing a water blank reagent control in place of DNA.

2. Paternal DNA can be tested to confirm VNTR/STR inheritance patterns when homozygous fragments (single band) are detected in the fetal-derived DNA.

3. When VNTR/STR analysis detects maternal DNA in the fetal I y-derived sample, the antigen status of the potentially compatible fetus is not confirmed unless the blood group genotype has been validated for a similar range of DNA mixtures. Alternatively, amniocytes can be cultured (Protocols 3.4 and 3.5) to confirm the fetal blood group antigen status.


1. The water blank must not have any visible amplified product.

2. Test and control samples will have no more than two amplified products of the approximate size as shown below.

Tandem repeat Nucleotide repeat Repeat size (bp)

Apo B 30 825

D1S80 16 530

D17S5 70 378

HR-1001/2 4 283

HR-1003/4 4 195

HR-1007/8 3 282

HR-1009/10 3 214

3. The amniotic fluid or cervical DNA is considered to be of fetal origin if at least one VNTR/STR shows a different sized fragment when compared with the maternal DNA.


1. The four STR sequences should be analysed to provide sufficient data for exclusion analysis.

2. Always electrophorese the maternal and fetally-derlved PCR amplified products in side-by-side lanes to assess VNTR/STR patterns for non-maternal origin.

3. Trinucleotide STR amplified sequences may show Taq polymerase-dependent amplification 'stutter' due to the variable addition of an adenine at the 3'-ends of the fragment.

4. The technique is used to confirm the origin of amniotic fluid or cervical smear DNA when a blood group compatible fetus is identified who is at risk for haemolytic disease of the newborn or neonatal alloimmune thrombocytopenia (see Denomme et al (1995) for further discussion).


3.17 Multiplex PCR Analysis Principle

Simultaneous amplification of sequences using a single PCR reaction. This technique relies on sequence-specific amplification primers to detect two or more target sequences. The absence of an amplification product indicates that the sequence differs at or near the end of either sequence-specific primer and suggests the presence of a variant allele.


Extracted DNA (see Protocol 3.6).

Equipment and Materials

Microcentrifuge Thermal cycler Micropipettors (general use) Micropipettors (PCR use only) Filter barrier micropipette tips

1.5 mL and 0.2 mL microcentrifuge tubes 10 x PCR buffer 25mM MgCI2

100 mM dNTPs (Promega, Cat #U1240) Nuclease-free ddH20 (Promega Corp, Cat #P119C) Allele-specific primers (100 ng/pL) Control sense/antisense primers (100 ng/pL) HotStarTaq DNA Polymerase (Qiagen Inc., Cat #203205)

Reagent Preparation

100 mM dNTPs are diluted to 10 mM using nuclease-free ddH20.


1. Select the sequence-specific primers that are appropriate for the polymorphism under analysis (see ABO or Rh Facts Sheets in Part 4).

2. Prepare a PCR cocktail (see below) for the total number of samples to be analysed. Make up a volume that is 10% greater than the total volume required that includes the samples, controls and water blank.

3. Add DNA, vortex and flash spin tubes.

4. Amplify according to the multiplex PCR profile below.

5. Combine 10 pL of the PCR product with 2 pL of 6 x loading buffer (see Protocol 3.12) and electropherese using an appropriate gel system (see Part 2 for type and concentration of gel).

6. Photograph the gel. If unexpected bands are found, refer to the troubleshooting checklist in Part 2 (Table 2.4).

Multiplex PCR cocktail

Reagent One sample _samples

Each of the sense primers 1.0 pL _pL

Each of the antisense primers 1.0 pL _pL

HotStarTaq polymerase (5.0 U/pL) 0.3 pL _pL

Sterile ddH20 26.7 pL _pL

Total 45.0 pL _pL

Note: water volume defined for 12 primers. Adjust water accordingly when changing number of primers.

Combine 45 pL of either cocktail with 5 pL of DNA (1 pg total) in a 0.2 mL microcentrifuge tube.

PCR profile

95°C for 15 minutes for HotStarTaq polymerase.

95°C for 60 seconds, 50 to 62°C for 60 seconds (see Procedural Note 3 below), 72°C for 45 seconds for 32 cycles. 72°C for 5 minutes, and 4°C hold.

Procedural Notes

1. The precautions listed in Protocol 3.8 will help avoid contamination.

2. The addition of Taq polymerase directly to the reaction mix along with DNA is possible for certain types of Taq polymerases. For example, the enzymatic activity of HotStarTaq polymerase is inhibited at room temperature but is activated upon heating above the annealing temperature. Therefore, mis-priming is avoided.

3. The PCR cocktail and profile are recommendations. The PCR conditions may need to be optimized (see Protocol 3.14).

Quality Control

1. The water blank (no DNA) must not have any bands.

2. Control DNA for each polymorphism must be analysed each time.


1. All tests and control samples must have at least one of the bands amplified or an internal control band to validate the assay.

2. The test sample should have the corresponding band if the allele is present.

3.18 Southern Analysis Principle

Genomic DNA is subjected to restriction enzyme digestion and size-separated by electrophoresis in an agarose gel. The DNA is transferred to a nylon membrane and analysed using digoxigenin (DIG)-11-dUTP labelled probes. Probes may be reverse transcribed cDNA, single exons, or larger regions of the gene. The hybridization of the DIG-labelled probe to the DNA is detected with anti-DIG/alkaline phosphatase and a chemi-luminescent substrate. Chemiluminescence is detected by exposure of the membrane to X-ray film.


Extracted DNA (Protocol 3.6).

Equipment and Materials

Microcentrifuge Thermal cycler

Hybridization oven and bottles

DNA transfer apparatus, membrane and blotting paper (Scheleicher & Scheull TurboBlotter and Nytran membrane - 15 cm x 20 cm, Cat #416318) UV cross-linker

X-ray cassette with intensifying screens (Eastman Kodak, Cat #KP 69065-E) X-OMAT AR film (Eastman Kodak, Cat #165-1454) Acetate page protectors Micropipettors PCR amplifed fragments: exon fragment exon-intron fragments 100 ng/pL primers for probe synthesis (for Rh analysis, see Part 4, Rh Facts Sheets)

DIG-11 -dUTP Probe Synthesis Kit (Boehringer Mannheim, Cat #1636090)

DIG Luminescent and Detection Kit (Boehringer Mannheim, Cat #1363514)

DIG Easy Hybridization Solution (Boehringer Mannheim, Cat #1603558) DIG Wash and Block Buffer Set (Boehringer Mannheim, Cat #1585762) Sodium dodecyl sulphate (SDS; Sigma, Cat #L-4509)

Reagent Preparation

250 mm HCI

Denaturation solution (0.5 n NaOH/1.5 m NaCI) Neutralization solution (0.5 m Tris-HCI, pH 7.5/3 m NaCI) 20 x SSC (3 m NaCl/300 mm sodium citrate, pH 7.0) 5 x SSC (1:4 (v:v) dilution of 20 x SSC in ddH20) 2 x Wash solution (2 x SSC containing 0.1% SDS) 0.5 x Wash solution (0.5 x SSC containing 0.1% SDS)


A. Probe synthesis

1. Follow the manufacturer's instructions to prepare a PCR cocktail for DIG probe labelling. Use 5 pL of a 1:200 dilution of the gel-purified fragment as the DNA template.

2. Amplify the probe according to the PCR profile used to obtain the initial PCR fragment with the number of cycles reduced to 30. Amplify the labelling control according to the manufacturer's instructions.

3. To verify probe amplification efficiency, combine 5 pL of the PCR product with 1 pL of 6 x loading buffer and electrophorese using a 3% agarose gel (Protocol 3.11).

4. Photograph the gel under UV light. The expected PCR products should be clearly visible.

B. Southern blotting

1. Digest 5 pg of genomic DNA with the restriction enzyme of interest, (e.g. Hae III, Msp I, Sph I) according to the instructions provided with the enzvme.

2. Prepare a 1% agarose gel (15 cm x 25 cm) in TBE (Protocol 3.11). Dilute the digested DNA in 6 x DNA loading buffer (Protocol 3.11) and electrophoresc until the bromophenol blue dye has migrated to within 5 cm of the bottom of the gel.

3. Place a fluorescent ruler next to the DNA ladder lane and photograph the gel under UV light.

4. Submerge the gel in denaturation solution twice for 15 minutes each. Rinse the gel with water.

5. Submerge the gel in neutralization solution twice for 15 minutes.

6. Prepare the nylon membrane for blotting according to the manufacturer's instructions.

7. Assemble the transblotting apparatus. Mark the position of the agarose lanes on the edge of the membrane using a pencil.

8. Transblot the DNA for a minimum of 6 hours in 20 x SSC.

9. Cross-link the DNA to the membrane using 2.4 x 105 millijoules of UV light. Rinse the membrane briefly in water and allow to air dry.

C. Membrane Hybridization

1. Place the membrane in a hybridization bottle containing 10 mL of DIG Easy Hybridization Solution and cap. Pre-hybridize at 37-42°C for 2 hours with rotation in a hybridization oven.

2. Heat the DIG-labelled probe at 100°C for 10 minutes to denature the DNA. Chill on crushed ice.

3. Prepare at least 3.5 mL of DIG Easy Hybridization Solution plus the DIG-labelled probe for each 100 cm2 membrane.

4. Discard the pre-hybridization solution. Add the DIG Easy Hybridization Solution containing the DIG-labelled probe (5-25 ng/mL). Allow the probe to hybridize overnight at 37-42°C.

5. Submerge the membrane in 2 x washing solution twice for 5 minutes each.

6. Submerge the membrane in 0.5 x washing solution twice for 5 minutes each.

D. Chemiluminescent detection of DIG-label

1. Equilibrate the membrane in DIG Wash for 1 minute.

2. Allow the chemiluminescent substrate to come to room temperature.

3. Submerge the membrane in DIG Block Buffer for 30-60 minutes.

4. Dilute the anti-DIG-alkaline phosphatase 1:10,000 (v:v) using the Block Buffer.

5. Submerge the membrane in the diluted antibody solution for 30 minutes.

6. Discard the antibody solution. Submerge the membrane in DIG Wash twice for 1 5 minutes each.

7. Discard the DIG Wash and equilibrate the membrane in Detection Buffer for 2 minutes.

8. Dilute the substrate reagent provided in the kit 1:100 (v:v) using the Detection Buffer.

9. Place the membrane between two sheets of acetate plastic page protectors. Gently lift the top sheet and add approximately 0.5 mL of substrate per 100 cm2 of membrane. Lower the top sheet of plastic. With a damp tissue, wipe the top sheet to remove any bubbles present under the sheet and to create a liquid seal around the membrane. Incubate for 5 minutes.

10. Transfer the semi-dry membrane to a plastic bag and seal the bag. Allow 7-8 hours for the chemiluminescence to reach steady state.

11. Incubate the membrane at 37°C for 15 minutes prior to exposure.

12. Expose the membrane to X-ray film. Multiple exposures from 20 minutes to 2 days are possible from a single blot.

Procedural Notes

A. Probe synthesis

1. See Protocol 3.8 for precautions to help avoid PCR contamination.

2. It is recommended to use a control template that can be DIG labelled (primers and template provided with the kit).

3. The semi-quantitative method (Protocol 3.7A) can be used to estimate the amount of DIG-labelled probe in place of the estimated yield using the spot test.

4. DIG-labelled DNA probes can be obtained also by random primary or nick translation.

B. Southern blotting

1. The photograph of the fluorescent ruler and agarose gel is used to determine the physical position of the DNA base pair ladder relative to the DNA fragments seen on the exposed X-ray film.

2. Do not allow the membrane to dry out until the DNA has been cross-linked.

C. Membrane hybridization

1. All incubations are at room temperature with shaking unless otherwise stated.

2. The DIG-labelled probe in the DIG Easy Hybridization Solution can be reused if desired. Store at -20°C for up to 1 year.

Quality Control

1. PCR contamination is detected by testing a water blank in place of the DNA template.

2. The chemiluminescent kits provide reagents to control for the DIG PCR labelling step.

3. Follow the manufacturer's instructions to estimate the yield of the DIG-labelled probe using a spot test and the DIG-labelled control.


DNA will vary in their Southern restriction maps depending on the zygosity and inherited polymorphisms for the gene of interest.


1. Common genotypes can be used to obtain restriction maps.

2. Mapping a gene locus using Southern analysis and many restriction enzymes can differentiate certain haplotypes and is a useful tool for the study of variants or antigen-negative phenotypes with a discrepant DNA-based genotype.


Ausubel et al (1997); Southern (1975).

3.19 References

Ausubel, F.M., Brent, R., Kingston, R.E., et al (1997) Current Protocols in Molecular Biology (3 volumes). John Wiley & Sons, New York.

Denomme, G. and Fernandes, B.J. (1999) The assignment of RHD gene zygosity by densitometric quantification. Abstract. Transfusion 39 (Suppl 1) 5IS.

Denomme, G.A., Waye, J.S., Burrows, R.F., et al (1995) The prenatal identification of fetal compatibility in neonatal alloimmune thrombocytopenia using amniotic fluid and variable number of tandem repeat (VNTR) analysis. Br J Haematol 91, 742-746.

Ellis, W.D., Mulvaney, B.D. and Saathoff, D.J. (1975) Leukocyte isolation by sedimentation: The effect of rouleau-promoting agents on leukocyte differential count. Prep Biochem 5,179-187.

Epstein, C.J. (1982) The use of growth factors in stimulate the proliferation of amniotic fluid cells. Methods Cell Biol 26, 269-276.

Hecht, F., Peakman, D.C., Kaiser-McCaw, B., et al (1981) Amniocyte clones for prenatal cytogenetics. Am ] Med Genet 10, 5 1-54.

Orita, M., Iwahana, H., Kanazawa, H., et al (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86, 2766-2770.

Orita, M., Suzuki, Y., Sekiya, T., et al (1989b) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874-879.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (3 volumes), 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503-517.

Tellez, A. and Rubinstein, P. (1970) Rapid method for separation of blood cells. Transfusion 10, 223-225.

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