METHODS

Samples

Twenty three peripheral blood specimens sent to the Mayo Clinic laboratory for haemoglobin testing were used in a non-blinded, retrospective analysis. The specimens had been stored at room temperature for variable amounts of time (range, 2–7 days) and 11 of the samples had been frozen (−20°C; range, 2–18 months) before use.

Whole genome amplification using MDA

The kit (REPLI-g 625s) from Molecular Staging Inc (New Haven, Connecticut, USA) was used according to the manufacturer’s instructions. Briefly, 1 μl of blood (approximately 5000 nucleated cells) was diluted in 34 μl of phosphate buffered saline, lysed by the addition of 35 μl of Fast Lysis solution, and neutralised by the addition of 35 μl of Lysis Stop buffer. A 2.5 μl aliquot of the lysis product was then mixed with 40 units (0.5 μl) of φ29 DNA polymerase, 12.5 μl of random primers and dNTPs, and 34.5 μl of distilled H₂O to a final volume of 50 μl and incubated at 30°C for 16 hours. In some cases, MDA was performed starting with genomic DNA isolated using column technology (see below). In these cases, the procedure was modified such that 1 ng of DNA was mixed directly with the enzyme and primers and incubated as described above.

Quantitative and qualitative analysis of the MDA products

The MDA products were quantitated using a fluorescence based method that is specific for double stranded DNA (PicoGreen^®; Molecular Probes, Eugene, Oregon, USA). Aliquots (5 μl) of the MDA product were also analysed using agarose gel electrophoresis (1.0% agarose, 100 V, 60 minutes, stained with ethidium bromide) and the products visualised using the Alpha Imager gel documentation system (San Leandro, California, USA).

DNA isolation using column technology

The Wizard genomic DNA purification kit (Promega, Madison, Wisconsin, USA) was used to isolate genomic DNA from 0.3 to 3 ml of whole, never frozen blood using the manufacturer’s instructions.

Sequencing of α and β haemoglobin genes

Table 1 shows the primers used for PCR amplification and subsequent sequencing of the α and β haemoglobin genes. PCR was performed in a 25 μl reaction volume containing 50 mmol/litre KCl, 10 mmol/litre Tris/HCl (pH 8.3), 200 umol/litre each dNTP, 1.5 mmol/litre MgCl₂, 0.2 μmol/litre each primer, 1.25 units of hot start Taq DNA polymerase, and 5 μl of Q-solution (US-Qiagen, Valencia, California, USA). The reactions were amplified in an MBS Satellite thermal cycler (Thermal Hybaid, Waltham, Massachusetts, USA) with an initial incubation at 95°C for 15 minutes, followed by 35 cycles of 94°C for 30 seconds and 61°C for one minute, with a single final incubation of 72°C for one minute. Sequencing was then performed using the dye terminator method with fragment analysis by capillary electrophoresis on the ABI 377 (Applied Biosystems, Foster City, California, USA) using Chromas 2.0 sequence analysis software (Technelysium Pty Ltd, Tewontia, Australia). Both strands of DNA in each specimen were sequenced.

RESULTS

Quantitation using the fluorescence based method revealed that each reaction, starting with either never frozen or previously frozen whole blood, produced approximately 50 μg of double stranded DNA (range, 44–59 μg; table 2). Because only approximately 2.4% of the DNA released during the cell lysis step was amplified (see methods), the theoretical total yield from the amplification of 1 μl of whole blood was estimated to be approximately 2 mg (table 2). Similar yields were obtained when the starting template was 1 ng of genomic DNA isolated using column technology. Specimen age before processing did not appear to have a significant affect on the yield. Qualitative analysis of the MDA products using agarose gel electrophoresis (fig 1) revealed that the amplified fragments were mostly greater than 20 kb and showed smaller average fragment size than the genomic DNA extracted using column technology (fig 1A). In addition, although the manufacturer’s recommendation for MDA incubation was 16 hours, as little as three hours of incubation was sufficient to produce a substantial amount of DNA (fig 1B).

In each sample, the DNA sequence from both strands showed a normal haemoglobin gene sequence in the entire region expected to be normal, and the expected mutations were correctly identified (table 3; fig 2). There was 100% concordance between the sequences obtained using MDA products as a template and those obtained using DNA extracted by the column method as a template.

DISCUSSION

Our results show that MDA is a useful method for amplifying very small amounts of genomic DNA before haemoglobin gene sequencing. Reliable results were obtained using whole blood specimens subjected to a variety of pretest conditions, including variable lengths of room temperature incubation, freezing, or previous genomic DNA extraction using a column based method. As little as 1 μl of whole blood or 1 ng of previously extracted DNA were adequate amounts of starting material, making the procedure useful for even the smallest clinical samples available.

Although no DNA purification was performed when whole blood was amplified, the small amount of residual cell debris did not hinder the sequencing application being tested and additional purification steps were not felt to be necessary. A previous report of MDA indicated that the method results in an average product length of 10 kb, with a range from 2 to 100 kb.³ Agarose gel analysis of the MDA products in our study indicated that the average DNA length obtained was probably longer (> 20 kb), but otherwise appeared consistent with predicted results. We also found that as little as three hours of amplification was sufficient to produce ample DNA for our purposes, which shortened the procedure time considerably, so that the entire amplification procedure required only approximately four hours (less than one hour of hands on technician time and three additional hours for incubation).

“We found that as little as three hours of amplification was sufficient to produce ample DNA for our purposes, which shortened the procedure time considerably”

We feel that our sequencing results were 100% specific because the mutations identified are all previously reported haemoglobin abnormalities, the same mutation was detected using sequencing of both DNA strands, the remainder of the sequence in each case showed a normal haemoglobin gene sequence, and there was 100% concordance with the results obtained using genomic DNA isolated by a standard column method as the sequencing template. Thus, our results indicate that the MDA technique is sufficiently reliable for amplification of peripheral blood samples before haemoglobin sequencing. Previous reports provide evidence to suggest that MDA is a robust and accurate technique, superior to PCR based genomic amplification methods.^{3– 5} The error rate of φ29 polymerase is estimated to be only 1/10⁶–10⁷, compared with approximately 3/10⁴ for Taq polymerase,⁶ and φ29 shows less amplification bias than Taq.³ Although our clinical test for globin gene sequencing uses PCR, errors caused by Taq polymerase do not appear to be frequent enough to confuse test interpretation. The main goal of our study was to verify the previous claims of φ29 enzyme fidelity in the context of our specific clinical needs.

Although we have only tested the MDA method in the specific clinical application of haemoglobin gene sequence analysis, we feel that MDA should be equally useful in a wide variety of clinical applications using small numbers of nucleated cells from essentially any source. However, our search of the English language literature revealed no previous reports of MDA applications for routine clinical testing.