__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.
- DNA cloning
- polymerase chain reaction, or PCR
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.
- DNA primers
- DNA pol
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.
- the protein would be denatured along with the DNA during the first heating step ad would have to be replaced after 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.
- PCR amplification
- PCR replication
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.