Overview

An important task for geneticists is to determine individuals' genotypes – genetic make-up. Most hereditary analysis requires some knowledge about the genotypes of closely related individuals. When reproduction is purely sexual (excluding clones, twins, and the like), individuals tend to differ from each other in genomic DNA sequence. In sexually reproducing species, each individual is genetically unique. Two kinds of individual differences in DNA sequence are nucleotide substitution and variation in sequence length.

This chapter describes a few of the many approaches to genotyping. One valuable method is determining the nucleotide sequence of DNA (DNA sequencing). Other, easier, more rapid methods provide partial information about genotypes. Four such methods are described, all based on polymerase chain reaction (PCR).

Sequencing DNA

Most DNA sequencing entails synthesis of DNA by the dideoxy method, which is based on the fact that 2′,3′-dideoxynucleotides terminate DNA synthesis. The deoxyribose of DNA lacks a hydroxyl group on the 2′ sugar; dideoxyribose lacks the 3′ hydroxyl group as well. Recall that DNA chain growth is 5′→3′; the α phosphate of the incoming nucleotide makes a covalent bond with the oxygen of the 3′-OH group of the growing polymer (Figure 28.1). If the 3′ nucleotide of the polymer lacks a 3′-OH group, then a nucleotide cannot be added and chain growth stops. A dideoxynucleotide can add to a growing polynucleotide chain, but, after it is incorporated into the polymer, it blocks further DNA synthesis.