Lambda Dna Amplification by Polymerase Chain Reaction (Pcr)
Lambda DNA Amplification by Polymerase Chain Reaction (PCR)
Introduction/ Background*
Since its introduction in 1985, polymerase chain reaction (PCR) has become a powerful tool in molecular genetic analysis. Today, it is used for applications such as cloning, analysis of DNA from ancient specimens, and analysis of human DNA for forensic applications. PCR is a test-tube DNA replication system for making many, many copies of, or amplifying, a defined segment of DNA. Using PCR, a selected target DNA can be amplified several million-fold in just a few hours. Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme-DNA polymerase-to amplify a specific segment of the genome.
During cellular DNA replication, enzymes first unwind and separate, or denature, the DNA double helix into single strands. Then, the enzyme RNA polymerase synthesizes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNA/RNA duplex acts as a "priming site" which allows the attachment of DNA polymerase. DNA polymerase then produces the complementary DNA strand by adding appropriate deoxyribonucleotides to the 3 ' end of the primer RNA. DNA polymerase absolutely requires a primer to begin DNA synthesis.
During PCR, high temperatures are used to denature the DNA molecule and separate it into single strands. In place of the short RNA primers used in cellular transcription, synthetic nucleotide sequences of single-stranded DNA (generally 20-30 nucleotides in length) are added to the mixture as primers. These synthetic DNA molecules are called oligonucleotides. When the temperature is lowered, these oligonucleotides anneal (form base pairs) with the single denatured strands of the DNA to be amplified. In the PCR reaction, two different primers are used. They are complementary to two regions of DNA that
Introduction/ Background*
Since its introduction in 1985, polymerase chain reaction (PCR) has become a powerful tool in molecular genetic analysis. Today, it is used for applications such as cloning, analysis of DNA from ancient specimens, and analysis of human DNA for forensic applications. PCR is a test-tube DNA replication system for making many, many copies of, or amplifying, a defined segment of DNA. Using PCR, a selected target DNA can be amplified several million-fold in just a few hours. Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme-DNA polymerase-to amplify a specific segment of the genome.
During cellular DNA replication, enzymes first unwind and separate, or denature, the DNA double helix into single strands. Then, the enzyme RNA polymerase synthesizes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNA/RNA duplex acts as a "priming site" which allows the attachment of DNA polymerase. DNA polymerase then produces the complementary DNA strand by adding appropriate deoxyribonucleotides to the 3 ' end of the primer RNA. DNA polymerase absolutely requires a primer to begin DNA synthesis.
During PCR, high temperatures are used to denature the DNA molecule and separate it into single strands. In place of the short RNA primers used in cellular transcription, synthetic nucleotide sequences of single-stranded DNA (generally 20-30 nucleotides in length) are added to the mixture as primers. These synthetic DNA molecules are called oligonucleotides. When the temperature is lowered, these oligonucleotides anneal (form base pairs) with the single denatured strands of the DNA to be amplified. In the PCR reaction, two different primers are used. They are complementary to two regions of DNA that
Cited: Freyer, Greg A., Micklos, David A., Crotty, David A. DNA Science: A First Course, 2nd ed. Cold Springs Harbor Laboratory Press, 2003. Freyer, G., Micklos, D. Laboratory DNA Science. Cold Springs Harbor Laboratory Press, 1996. Carolina Biological Supply Company, 2004