Rapid Isolation of PCR-Ready DNA for Prenatal Diagnosis Using the MasterAmp™ Buccal Swab DNA Extraction Solution, EPICENTRE® Biotechnologies

EPICENTRE Forum 4 (4)

|Rapid Isolation of PCR-Ready DNA for Prenatal Diagnosis Using the MasterAmp™ Buccal Swab DNA Extraction Solution|

Marlena Lewandowska-Skarbek and Jeanne Bourdeau-Heller, University of Wisconsin State Laboratory of Hygiene, Madison, WI 53706

Traditionally, the source of high-quality DNA for the prenatal diagnosis of genetic disorders has been chorionic villi samples, fetal blood, or amniotic fluid. Adequate amounts of DNA can be extracted from amniotic fluid cells beginning at 8 weeks gestation, and these samples are suitable for prenatal diagnosis using PCR.¹ Because in many cases the number of cells available for DNA isolation and diagnosis is limited, we were looking for a DNA extraction system that would produce sufficient genomic DNA for diagnostic purposes even with a limited amount of cells. The MasterAmp™ Buccal Swab DNA Extraction Solution (Epicentre Technologies) attracted our attention because of its simplicity, rapidity, and high DNA yields. Although this product was developed for DNA extraction from buccal cells,² we wanted to know how it would perform with other cell types. We demonstrate here that using the MasterAmp Buccal Swab DNA Extraction Solution, genomic DNA can be quickly isolated from chorionic villi samples and amniotic fluid cells, and the isolated DNA is of sufficient quality for PCR amplification. To verify the presence of DNA that could be reliably amplified, we performed PCR with primers used in the diagnosis of Fragile X mutations at the FRAXA and FRAXE loci as a screen for normal alleles, and with primers for the human ß-actin gene.

Sample preparation

The MasterAmp Buccal Swab DNA Extraction Solution was used to isolate DNA from the cell pellets of 15 cultured amniotic fluid samples and 10 cultured chorionic villi samples. The gestational ages of the samples ranged from 10 to 23 weeks (amniotic fluid) and 10 to 18 weeks (chorionic villi). Cells from the chorionic villi samples were collected from T-25 flasks (50-100% confluent) and amniotic cells were collected by centrifugation of 5 ml of amniotic fluid. The cell pellets were resuspended in 75 µl of Hank's Balanced Salt Solution (HBSS). One-third of the resuspended cells was used for DNA extraction using the protocol provided with the product, modified only for the cell collection steps (Table 1).³

Table 1. Modified MasterAmp Buccal Swab DNA Extraction Solution Protocol for Amniotic Fluid and Chorionic Villi Samples.

Add 25 µl of the resuspended cells to 500 µl of MasterAmp Buccal Swab DNA Extraction Solution.
Vortex mix for 10 seconds. Incubate the tube at 60°C for 30 minutes.
Vortex mix for 15 seconds.
Transfer the tube to 98°C and incubate for 8 minutes.
Vortex mix for 15 seconds.
Return the tube to 98°C and incubate an additional 8 minutes.
Vortex mix for 15 seconds. Chill the tube on ice long enough to reduce the temperature.
Pellet cellular debris by centrifugation at 4°C for 5 minutes.
Carefully transfer the supernatant containing the DNA to a clean tube without including any of the pelleted cellular debris.

PCR amplification

The DNA samples were used for Fragile X mutation screening of the FRAXA and FRAXE loci, and for amplification of the ß-actin gene. PCR-based screening, which allows simultaneous amplification of the triplet repeat sequences at the FRAXA and FRAXE loci, is a rapid and inexpensive approach to detect alleles of normal size. Since the majority of referrals for the Fragile X syndrome are negative, only a few samples must then be tested by Southern blotting following PCR screening.

For amplification of the FRAXA CGG repeat, 20 pmoles of each primer (FXD and FXE) were used, and for amplification of the FRAXE CCG repeat, 35 pmoles of primers 598 and 603 were used. The primer sequences were:
FXD: 5'-TGACGGAGGCGCCGCTGCCAGGGGGCGTGC-3'
FXE: 5'-GAGAGGTGGGCTGCGGGCGCTCGAGGCCCA-3'
598: 5'-GCGAGGAAGCGGCGGCAGTGGCACTGGG-3'
603: 5'-CCTGTGAGTGTGTAAGTGTGTGATGCTGCCG-3'

We used a modified amplification method of Wang et al.⁴ to generate a FRAXA PCR product 74-224 bp in length (corresponding to a normal range of 2-54 FRAXA CGG repeats) and a FRAXE product with a minimum size of 306 bp (for an allele with six FRAXE CCG repeats). Only 5 µl of the 350 µl of DNA extracted from cultured amniotic fluid or chorionic villi were used for each 50 µl PCR amplification. Initial amplification to determine optimal PCR conditions was performed using the MasterAmp PCR Optimization Kit (Epicentre) and 2.5 units of AmpliTherm™ DNA Polymerase (Epicentre) (Figure 1). Then, amplification was performed with MasterAmp 2X PCR PreMix D (100 mM Tris-HCI, pH 8.3, 100 mM KCI, 400 µM each dNTP, 3.0 mM MgCl₂, and 4X MasterAmp PCR Enhancer*), which we determined to be the optimal PreMix for duplex PCR of the Fragile X mutations.

Figure 1. Optimization of amplification of the Fragile X mutations at the FRAXA and FRAXE loci. DNA isolated from a single cultured amniotic fluid sample was amplified using the MasterAmp PCR PreMixes A-J as described. 25 µl of each reaction were resolved in a 2% agarose gel and visualized with ethidium bromide staining. Cycling conditions were: 37°C for 4 minutes, 95°C for 6 minutes, followed by 30 cycles at 95°C for 1 minute, 61°C for 90 seconds, 72°C for 2 minutes, followed by a final extension step at 72°C for
5 minutes.

All extracted DNA samples (32 total) amplified well. Five samples from each group (DNA from cultured amniotic fluid and from cultured chorionic villi) show the presence of the normal allele for FRAXA and FRAXE loci (Figure 2). The presence of two bands in the correct size range in a male, or heterozygosity for alleles of normal size in a female, excludes the presence of an expansion mutation at both loci.

Figure 2. Amplification of a normal allele at the FRAXA and FRAXE loci using DNA from amniotic fluid and chorionic villi isolated using the MasterAmp Buccal Swab DNA Extraction Solution. Lane 1 in each panel, 50 bp ladder; Panel A, Lanes 2-6, DNA isolated from cultured amniotic fluid (AF); Panel B, Lanes 2-6, DNA isolated from cultured chorionic villi (CV). The lower molecular weight band corresponds to FRAXA normal allele and the upper band to the normal allele of FRAXE.

Figure 3 shows amplification of three samples from each cell type using primers that generate an 850 bp target within the ß-actin gene of human DNA. The sequences of the primers used were: 5'-AGCGGGAAATCGTGCGTGACA-3' and 5'-GTGGACTTGGGAGAGGACTGG-3'.

Figure 3. Amplification of the human ß-actin gene. Lane 1, 50 bp ladder; Lanes 2-4, DNA isolated from cultured amniotic fluid (AF); Lanes 5-7, DNA isolated from cultured chorionic villi (CV).

The MasterAmp Buccal Swab DNA Extraction Solution can be used for the extraction of DNA from different sources. We have demonstrated that we were able to obtain high-quality, PCR-ready DNA from both cultured amniotic fluid and cultured chorionic villi using the MasterAmp Buccal Swab DNA Extraction Solution. We were also able to obtain similar results using uncultured amniotic fluid cells (data not shown). The high DNA yields obtained using this procedure allowed us to use only 5 µl of the final 350 µl supernatant in each of our amplification reactions. Because this is a very simple and rapid method to obtain human genomic DNA suitable for PCR amplification, we now routinely use the MasterAmp Buccal Swab DNA Extraction Solution for DNA sample preparation for use in molecular diagnosis. The extracted DNA can be used for PCR amplification, and the presence of MasterAmp PCR Enhancer in the Extraction Solution increases amplification efficiency.

Rebello, M.T. et al. (1991) Prenatal Diagnosis 11, 41.
Watson, J. (1997) Epicentre Forum 4 (1), 6.
MasterAmp™ Buccal Swab DNA Extraction Solution Product Information Sheet, 1997.
Wang, Q. et al. (1995) J. Med. Genet. 32, 170.

MasterAmp™ Buccal Swab DNA Extraction Kit