Lecture 5 Flashcards
(5 cards)
PCR
The polymerase chain reaction (PCR) is a technique that enable rapid amplification of a specific segment of DNA into millions to billions of copies (complete or partial) from only a small amount of starting material (i.e., DNA template or target sequence).
PCR amplify DNA fragments of between 0.1 and 10 kb in length, although some techniques allow for amplification of fragments up to 40 kbp.
Components
- DNA template that contains the DNA target region to amplify
- DNA polymerase
- two DNA primers that are complementary to the 3’ ends of each of the sense and anti-sense strands of the DNA target
- deoxynucleoside triphosphates, or dNTPs (each of the 4 nucleotides containing triphosphate groups)
- a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
- bivalent cations, typically magnesium (Mg) or manganese (Mn) ions; Mg2+ is the most common, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn2+concentration increases the error rate during DNA synthesis.
- monovalent cations, typically potassium (K) ions
Steps PCR
PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles.The individual steps common to most PCR methods are as follows:
*Initialization - required for DNA polymerases that require heat activation (hot-start PCR), at 94–98°C.
*Denaturation - 94–98°C for 20–30 seconds for denaturation of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
*Annealing: 50–65°C for 20–45 seconds, allowing annealing of the primers to each of the single-stranded DNA templates.
*Extension/elongation: 75–80°C (167–176°F) In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture.
*Final elongation: optional at 70–74°C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
*Final hold: at 4–15°C for an indefinite time, may be employed for short-term storage of the PCR products.
Stages
PCR process can further be divided into three stages based on reaction progress:
*Exponential amplification: The replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions. At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. The reaction is very sensitive: only minute quantities of DNA must be present.
*Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
*Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
Research applications
Research applicationsPCR has been applied to many areas of research in molecular genetics:
*DNA cloning.
*PCR-based analysis of DNA from ancient sources
*Gene expression by quantitative PCR to quantitate the actual levels of expression
*DNA modifications
*Site-directed mutagenesis
