The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway. A DNA probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples. Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured. Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target. Thus the labeled probe becomes specifically associated with the target. The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signaled by the bound label.
The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix. These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.
The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix. These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.
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