3.3.1 POLYMERASE CHAIN REACTION
The Polymerase Chain Reaction (PCR) is the most common DNA amplification method in molecular biology, it was invented by Kary Mullis while working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. His invention won him a Nobel Prize in Chemistry in 1993. PCR has revolutionized the field of molecular biology as it has enabled researchers to perform experiments easily that previously had been unthinkable. Before the mid-1980s, when PCR was developed, molecular biologists had to use laborious and time-consuming methods to identify, clone, and purify DNA sequences they wanted to study.
PCR uses a pair of primers to span a target region in template DNA, and then polymerizes …show more content…
Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA sequences prior to molecular cloning. In DNA cloning the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO) (Brown and Brown, 2015). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA …show more content…
coli extract to produce a precipitate that contained the cellular nucleic acids and a nucleic acid-free supernatant. Streptomycin sulfate was used frequently at that time to remove nucleic acids, often a hindrance to protein purification in bacterial extracts. Assay of the nucleic acid-free supernatant (S-fraction) and the nucleic acid-containing precipitate (P-fraction) showed them to be devoid of dTTP incorporation activity. However, when the two fractions were combined, activity was restored. Kornberg and his team observed that prior incubation of the extract or the P-fraction for a few minutes at 37 °C substantially increased activity. Clearly more than one enzyme was required for the incorporation of dTTP into an acid-insoluble product. The complexity of the system became even more apparent when they began to fractionate S and P. The P-fraction could be sub-fractionated into two fractions, one heat-labile and the other heat-stable, both of which (in combination with the S-fraction) were necessary for activity. The S-fraction could be separated into a heat-labile fraction and a heat-stable fraction that could pass through a dialysis membrane, i.e. was dialyzable. The latter could be further fractionated by Dowex-1 chromatography into three discrete fractions. (Dowex-1, an anion exchange resin used at the time, separated low molecular weight acidic compounds.) Thus, incorporation of dTTP
The Polymerase Chain Reaction (PCR) is the most common DNA amplification method in molecular biology, it was invented by Kary Mullis while working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. His invention won him a Nobel Prize in Chemistry in 1993. PCR has revolutionized the field of molecular biology as it has enabled researchers to perform experiments easily that previously had been unthinkable. Before the mid-1980s, when PCR was developed, molecular biologists had to use laborious and time-consuming methods to identify, clone, and purify DNA sequences they wanted to study.
PCR uses a pair of primers to span a target region in template DNA, and then polymerizes …show more content…
Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA sequences prior to molecular cloning. In DNA cloning the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO) (Brown and Brown, 2015). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA …show more content…
coli extract to produce a precipitate that contained the cellular nucleic acids and a nucleic acid-free supernatant. Streptomycin sulfate was used frequently at that time to remove nucleic acids, often a hindrance to protein purification in bacterial extracts. Assay of the nucleic acid-free supernatant (S-fraction) and the nucleic acid-containing precipitate (P-fraction) showed them to be devoid of dTTP incorporation activity. However, when the two fractions were combined, activity was restored. Kornberg and his team observed that prior incubation of the extract or the P-fraction for a few minutes at 37 °C substantially increased activity. Clearly more than one enzyme was required for the incorporation of dTTP into an acid-insoluble product. The complexity of the system became even more apparent when they began to fractionate S and P. The P-fraction could be sub-fractionated into two fractions, one heat-labile and the other heat-stable, both of which (in combination with the S-fraction) were necessary for activity. The S-fraction could be separated into a heat-labile fraction and a heat-stable fraction that could pass through a dialysis membrane, i.e. was dialyzable. The latter could be further fractionated by Dowex-1 chromatography into three discrete fractions. (Dowex-1, an anion exchange resin used at the time, separated low molecular weight acidic compounds.) Thus, incorporation of dTTP