DNA Sequencing

Prior to the performance of a sequencing reaction, the DNA template to be sequenced must be properly purified and quantitated. A sequence-specific complementary primer must be synthesized for use in the sequencing reaction. Sequencing primers must also be high-performance liquid chromatography (HPLC) purified or, at the very least, desalted. Samples must be purified following the sequencing reaction to rid the sample of salts, excess dyes, and excess primers that would compete for injection into the capillary. There are numerous protocols and commercial kits available for the postreaction purification. Ethanol precipitation is the least expensive purification method, but numerous gel-filtration and spin-column methods also can be used.

The ability to sequence DNA has been essential to the field of molecular pathology because sequence information is a prerequisite for PCR, PCR alternatives, and hybridization with probes necessary for successful Southern blot analysis. The method for DNA sequencing developed by Sanger, Nicklen, and Coulson6 is the basis for most DNA sequencing performed both in clinical laboratories and for the Human Genome Project.

The Sanger sequencing reaction uses a single DNA primer and DNA polymerase with linear amplification rather than the exponential amplification of PCR (see below). Components essential to the Sanger sequencing reaction include (1) an electrophoresis technique capable of clearly distinguishing single nucleotide length differences in DNA strands dozens or hundreds of nucleotides in length, (2) sequence-specific complementary primers, with one primer used in the forward reaction and the other used in the reverse reaction for each DNA template strand, and (3) the addition of small proportions of dideoxynu-cleoside triphosphates (ddNTPs) in addition to the conventional deoxyribonucleoside triphosphates (dNTPs) used in the polymerase reaction. Dideoxynucleotides differ from deoxynucleotides by having a hydrogen atom attached to the 3' carbon rather than an OH group, which is present on the deoxynucleotide. Because the ddNTPs lack a 3'-hydroxyl group, elongation of the newly polymerized DNA chain cannot occur once a ddNTP has been incorporated (arabinonucleosides can also be used as inhibitors of elongation). The end result is a set of newly synthesized DNA chains that are complementary to the template DNA but that vary in length, with the length determined by the point at which the ddNTP was incorporated into the 3' end of the chain.

In the original paper,for each template four reactions were performed with the addition of a single inhibitor to each, either ddGTP, ddATP, ddTTP or araCTP.6 The DNA chains were separated by polyacrylamide gel electrophoresis under denaturing conditions and visualized using (a-32P)-dATP on a radio-autograph. These reactions were run in consecutive lanes of the gel, and the complementary DNA sequence was determined by manual inspection based on the size of each chain, and the specific ddNTP in the reaction.

Today, sequencing is very similar except that radioactive labeling has largely been replaced by fluorescent labeling. Two major categories of fluorescent labeling exist. In dye-primer labeling, the sequencing primer is labeled, and the sequencing reaction requires 4 tubes differing only in the incorporation of the specific ddNTP. In dye-terminator labeling, the sequencing primer is unlabeled and instead each ddNTP is labeled with a different fluorophore. This enables the entire sequencing reaction to be performed in a single tube. Dye-terminator labeling has therefore taken precedence in sequencing. Dye-primer labeling is frequently used in fragment analysis for detection of microsatellite instability, loss of heterozygosity, forensic identification, or allogeneic bone marrow transplantation monitoring using short tandem repeat polymorphisms. Automated sequencers recognize both the size of the DNA chain and the fluorescent color of the chain to assign the nucleotide sequence, and also function as precise detectors for fragment analysis.7

Conventional DNA sequencing with polyacrylamide gel electrophoresis (whether using manual or automated sequence detection) is time-consuming and laborintensive. Therefore, the introduction of CE was a welcome change that facilitated the incorporation of sequencing and fragment analysis into the clinical laboratory.8 The sequencing reaction products must be purified before injection into the CE unit to remove excess salts, dyes, and unincorporated primers that would compete for injection into the capillary. Purification is accomplished by ethanol precipitation or a chaotropic salt-silica column technique. After the postreaction purification step, samples are then resuspended in a sample loading solution containing high-quality deionized formamide to denature the DNA. Formamide also provides a very stable environment for fluorescent dyes. The fragments are injected into the capillary, detected by laser-induced florescence, and rendered into sequence by the analysis software. An electropherogram of the DNA sequence is generated by the detection software by correlating the fluorescent intensity of each dye wavelength corresponding to a specific ddNTP as a function of migration time.

Examples of Applications of DNA Sequencing

1. Congenital adrenal hyperplasia mutation analysis

2. Retinoblastoma mutation analysis

3. BRCA1 mutation analysis

4. HIV genotyping to monitor drug resistance and sensitivity

5. High-resolution human leukocyte antigen (HLA)

typing for allogeneic bone marrow transplantation

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