PCR

__in cells remains the best method for preparing large quantities of a particular gene or other DNA sequence.
However, when the source of DNA is scanty or impure, the__ is quicker and more selective.

In this technique, any specific target segment within one or many DNA molecules can be quickly amplified in a test tube.
· With __, __can make billions of copies of a target segment of DNA in a few hrs, faster than the days it’d take to obtain the same number by screening a DNA library for a clone with the desired gene and letting it replicate within host cells.

__is being used increasingly to make enough of a specific DNA fragment to insert it directly into a __, entirely skipping the steps of making and screening a library.

In the __procedure, a __-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
· During each cycle, the reaction mixture is heated to __(separate) the DNA strands
· Then cooled to allow __(hydrogen bonding) of short, single- stranded __complementary to sequences on opposite strands at each end of the target sequence;
· finally a heat-stable __extends the primers in the 5’->3’ direction.

What would happen if a standard DNA pol were used?
The key to automating __was the discovery of an unusual heat-stable DNA pol, first isolated from cells of a bacterial species living in hot springs that could withstand the heat at the start of each cycle.

Just as impressive as the speed of PCR is its __.
· Only minute amounts of DNA need be present in the starting material, and this DNA can be in a partially degraded state, as long as a few molecules contain the complete target sequence.
· The key to this high specificity is the __, which hydrogen bond only to sequences at opposite ends of the target segment. (For high specificity, the primers must be at least __nucleotides long.)

By the end of the __cycle, __of the molecules are identical to the target segment, with both strands the appropriate length.
· With each successive cycle, the number of target segment molecules of the correct length doubles, soon greatly outnumbering all other DNA molecules in the reaction.

Despite its speed and specificity, __ cannot substitute for gene cloning in cells when large amounts of a gene are desired. Occasional errors during __ impose limits on the number of good copies that can be made by this method.

When __is used to provide the specific DNA fragment for __, the resulting clones are sequenced to select clones with error-free inserts.
Devised in 1985, __has had a major impact on biological research and biotechnology.

__has been used to amplify DNA from a wide variety of sources: fragments of ancient DNA from frozen wooly-mammoths; DNA from fingerprints or from tiny amounts of blood, tissue or semen found at crime scenes; DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders and DNA of viral genes from cells infected with viruses that are difficult to detect, like HIV.