Diagnostic Techniques

This work shows that single sequencing reactions analyzed on an automated DNA sequencer can be an efficient way of screening PCR products for known mutations. We have analyzed a mutation in exon 10 of the human aromatase gene and show that an unambiguous genotype could be elucidated in more than 90% of the analyzed samples. Compared to analysis by full sequencing, 4 times more samples can be analyzed per gel, so that the sample capacity of the gel is approaching that of alternative gel-based methods for genotype analysis. Unlike many of these, the method offers direct identification of the variant sequence position and on-line analysis without the need of postelectrophoretic processing of the gel. INTRODUCTION Of the many methods available for mutation detection, DNA sequencing of polymerase chain reaction (PCR) products from the region of interest is the most informative; however, it is also among the most labor-intensive (for recent reviews see References 1 and 2). The advantage of DNA sequencing is that mutations in the sequenced DNA are unambiguously detected whether they are known beforehand or not. The drawback of sequencing, compared with other gel-based methods such as restriction fragment-length polymorphism (RFLP) analysis, single-strand conformation polymorphism (SSCP) analysis and others, is mainly that fewer samples can be analyzed per gel. This is especially true for manual, radioactive sequencing and for DNA sequencers based on one-dye sequencing, where four gel lanes are needed for each sample to be analyzed. Analysis of sequencing reactions on amplified genomic DNA can identify not only individuals that are homozygous for a given allele but also heterozygotes. When the amplified DNA consists of DNA from two different alleles, both alleles will produce sequencing ladders corresponding to their sequences, and the sequence read from the gel will be a composite of the two different sequences. A point mutation in one allele will then be seen by the presence of signal in two lanes in this position. In elucidating the two separate sequences from such data, the use of T7 DNA polymerase as the sequencing enzyme has been extremely helpful, especially when using biotin-labeled PCR products (3), fluorescence-labeled sequencing primers and automated DNA sequencers. This is because this enzyme under proper reaction conditions does not discriminate between normal deoxyribonucleoside triphosphates and the corresponding dideoxy analogues (7,9). This leads to even peak intensities in the sequencing reactions, making it possible to identify heterozygosity in a given position not only from the presence of a signal in two of the four sequencing reactions, but also because these two peaks will be one half the size of their neighboring single peaks (4). The performance of T7 DNA polymerase in this respect is matched by the recently developed Taq DNA polymerase variant with a point mutation in the active site (F667Y), making it accept dideoxynucleotides far more readily than wildtype Taq DNA polymerase (8). In many circumstances, one is not interested in complete DNA sequences but only in differences between them. In these cases, single sequencing reactions have been used for many years, (e.g., to screen recombinant vectors for a given insert and for other purposes). In this work, we show that single sequencing reactions performed on PCR products, with subsequent analysis on an ALFexpress DNA Sequencer, can be as informative as complete sequencing reactions for the genotyping of known point mutations in human DNA. This is achieved by using a mutated Taq 832 BioTechniques High-Throughput Screening for Known Mutations by Automated Analysis of Single Sequencing Reactions BioTechniques 24:832-835 (May 1998) DNA polymerase, as described above, in cycle-sequencing reactions with one single dideoxyanalogue, chosen to correspond to the base found in one of the two alleles. In the example shown here, there is either a C or a T in a given position in the DNA. In a T-track sequencing reaction, a PCR product from a T/T individual will have a normally sized T peak in this position (contribution to the signal from both alleles), whereas a C/T genotype will have a T peak with one half the normal size (contribution from only one allele) and the sequence from a C/C individual will have no signal in the relevant position in the T track. This simplified sequencing-based genotyping method makes it possible to analyze four times the number of samples for each run on the sequencing gel without loss of sensitivity and accuracy. MATERIALS AND METHODS Part of the human aromatase gene (CYP19) was amplified from leukocyte DNA from 205 individuals by using primers that amplify 367 bp of exon 10 (6). PCR products were purified using Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA). Complete or T-track cycle sequencing reactions were performed using a Thermo Sequenase kit (Amersham Pharmacia Biotech, Little Chalfont, Bucks, England, UK) and a Cy5-labeled, fluorescent nested primer, ACATGCTGGAAATGATCTTT. The polymorphic site to be investigated was situated 75 nucleotides from the 5′ end of the primer. PCR product (0.5 μL containing approximately 0.1 μg DNA) was mixed with 0.5 pmol labeled primer in a total volume of 5 μL, after which 2 μL T termination mixture were added and thermal cycling initiated. One cycle consisted of 30 s at 95°C and 30 s at 50°C, and was repeated 25 times in a Model PTC100 Thermal Cycler (MJ Research, Watertown, MA, USA). After denaturation with formamide, 5-μL aliquots of the reactions were loaded into separate wells in a 6% polyacrylamide gel (acrylamide:bis 19:1; Saveen Biotech, Malmö, Sweden) containing 1× TBE (0.089 M Tris-base, 0.089 M boris acid, 2 mM EDTA disodium salt) and 7 M urea (Life Technologies, Gaithersburg, MD, USA) and fractionated by electrophoresis at 25 W in an ALFexpress DNA Sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden). After data collection, complete sequencing reactions were analyzed using the ALFwin software package, while single T tracks were analyzed using the AlleleLinks package (both from Amersham Pharmacia Biotech). Single-track analyses consisted of first identifying peaks, then sizing the products using two of the invariant sites (positions 64 and 80 from the 5′ end of the primer) as internal standards and then measuring the relative area under the relevant peaks by setting the area under one of the invariant T peaks neighboring the variant site to 1. The peak table was then exported to a text file and imported into Microsoft Excel (Microsoft, Redmond, WA, USA) for further analysis.