In 1961, researchers Sol Spiegelman and Edward Hall discovered that single-stranded DNA forms hydrogen bonds with a complementary strand of RNA and yields a double-stranded DNA-RNA molecule. For the next twenty years, DNA technologists changed the principle slightly and applied it to a DNA-DNA match. They researched the possibility of using a DNA strand to recognize a complementary DNA strand amid a mixture of other DNA strands, much as a key might seek out a lock. Eventually, they succeeded and brought forth the significant factor in DNA-based analyses: a molecule of DNA called a DNA probe.

A DNA probe is a relatively small (a few thousand bases in length), single-stranded molecule of DNA that recognizes and binds to a complementary segment of DNA on a large DNA molecule, as shown in figure below. Because DNA bases always pair A to T and G to C, a DNA probe interacts very specifically with nucleic acid sequences in target molecules of DNA. Like a left hand seeking one specific right hand, the probe DNA mingles among a mixture of DNA strands until it locates one Strand (or section of a strand) that is complementary. It then binds to that strand (or section).

The activity of a DNA probe is intimately linked to the biochemistry of DNA. A double-stranded DNA molecule tends to unwind and disassemble under certain laboratory conditions. These conditions include heating at a temperature over 90~ or subjecting the DNA to a pH higher than 10.5 or to organic compounds such as urea or formaldehyde. When so exposed, the hydrogen bonds between base pairs break and the complementary strands come apart. Such a process is called denaturation.

Denaturation can also be reversed. If the proper laboratory conditions of salt, temperature, and pH are established, the two single-stranded DNA molecules will reassemble to form the original duplex. This process is called hybridization (or renaturation) and is central to the use of the DNA probe. Under carefully controlled conditions, stable DNA duplexes will form only when complementary base pairing occurs perfectly along the entire length of the DNA strands.

With current technology, a DNA probe can be developed to detect virtually any nucleic acid sequence. It is not even necessary to know the actual sequence of bases in the target DNA. For example, the DNA can be encouraged to form complementary messenger RNA (mRNA) molecules. The mRNA molecules can then be isolated and combined with the enzyme reverse transcriptase plus appropriate building block nucleotides and other materials. Reverse transcriptase will use the isolated mRNA as a template and synthesize a complementary molecule of DNA (called cDNA). This new DNA is identical to the portion of DNA (the exons) functioning in protein synthesis at one particular time. The new DNA molecule is now used as a probe to detect the nuclear DNA whenever an identification is required.

A DNA probe can be as short as ten bases or greater than 10,000 bases in length. Clearly, the probe must be able to bind to (or "hybridize to") the target DNA, but it must also avoid hybridizing to other nucleic acid molecules that may be present. And once the probe-target molecule has formed, the combination must be stable.

During the early 1980s, the optimism generated by the use of DNA probes was counterbalanced by the problem of not having enough DNA to perform a reliable test. With insufficient target DNA, the probe's radioactive signal did not work well. Thus, it was important to amplify the target DNA. The technology for DNA amplification was developed in a procedure called the polymerase chain reaction, as we will see next.