Polymerase Chain Reaction Restriction Fragment Length Polymorphism One of

the simplest methods to genotype test samples is through restriction fragment length polymorphism (RFLP) analysis. With this method, after PCR, the PCR products are digested with an appropriate restriction enzyme and visualized by staining the gel after electrophoresis. If the test sample contains a genetic polymorphism that causes a gain or loss of the restriction site, then that sample will display a different migration pattern on the gel. Appropriate

Addition cf extension primer which will anneal immediately adjacent to (but not including) the polymorphic site

PCR cf area that includes polymorphic site

Denaturation

Addition cf extension primer which will anneal immediately adjacent to (but not including) the polymorphic site

Figure 12.2. Single base primer extension. This patient is heterozygous for this A to C substitution. The labels from both ddNTPs can be detected in this sample. In the case of a homozygous genotype, only one of the labels would be detected. Detection might be through ELISA, fluorescence, FRET, or FP.

Single stranded pcr products

Annealing cf extension primer

Extension by one base

Detection

Figure 12.2. Single base primer extension. This patient is heterozygous for this A to C substitution. The labels from both ddNTPs can be detected in this sample. In the case of a homozygous genotype, only one of the labels would be detected. Detection might be through ELISA, fluorescence, FRET, or FP.

quality control measures require that positive and negative controls be present in each assay to confirm that the restriction enzyme is active.

The main advantages of RFLP analysis are that it is simple to develop and use. It also does not require expensive equipment, and it is effective for genotyping a small number of samples. The principle disadvantages are that it is time consuming, labor intensive, and not amenable for large numbers of samples or genotypes. Another drawback is that not all SNPs produce a usable restriction site.

3.2.7.2 Single Base Primer Extension. One of the most effective ways to detecting polymorphism-is a technique known as singlebase primer extension (Fig. 12.2). In this simple assay, a region containing the polymorphism of interest is first amplified in a PCR reaction. The double-stranded PCR product is prepared into a single-stranded DNA fragment by heat or enzymatic digestion. A third primer is then used in the primer extension reaction. On the single-stranded PCR product, this third primer, called the primer extension primer, anneals immediately adjacent to, but only including, the site of the known mutation. After addition of DNA polymerase and labeled chain-terminating dideoxynucleotides corresponding to the wild-type and variant sequence, one of the two dideoxynucleotides will be added onto the primer at the site of the mutation. Unlike regular deoxynucleotides, which will extend indefinitely, dideoxynucle-

otides will only extend by one base. After completion of the primer extension reaction, detection of the reaction products may occur in any number of ways (13, 14).

An important distinguishing feature of primer extension is that assay specificity arises from the enzymatic specificity of DNA polymerase, not from the hybridization of the extension primer. Consequently, single base extension is rapidly gaining acceptance as the reaction biochemistry of choice for high-throughput genotyping of SNPs. It is well suited for high-throughput applications because the reactions can occur under similar reaction conditions, assay design and optimization are minimal, and it is portable to a variety of detection platforms (15). For these reasons, single-base primer extension is being licensed and adapted for use of a variety of different platforms and detection systems, including indirect colorimetric detection on ELISA-style microtiter plates (Orchid BioSciences' SNPStream 25K and SNPware 96 kits), DNA array product capture systems (SNPcode kits for use on Affymetrix's GeneChip and GeneFlex; Aper's APEX), fluorescent bead-based sorting devices (Luminex's LabMAP), capillary DNA sequencers (ABI's SnaPshot and Amersham Pharmacia Biotech's SNuPe), mass spectrometry (Seque-nom), and fluorescence polarization (Perkin Elmer).

3.2.1.3 Pyrosequencing. Another enzymatic genotyping method is pyrosequencing (Pyrosequencing AB). In this method, primer extension is monitored by luminometric detection of pyrophosphate, which is released on the addition of deoxynucleotides. The pyrophosphate is converted to ATP, which in turn stimulates luciferase to produce light which can be detected. As the deoxynucleotides are added, the complementary DNA strand is built up and the nucleotide sequence can be determined from the signal strength. Using pyrosequencing, the sequence of short 30-50 bp stretches of DNA can be determined (15,16). This method available in a 96-well format and might be useful for small low-throughput applications, but the high cost may be an issue when high genotyping throughput is necessary.

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