Lecture 18 - DNA technology 2 Flashcards
DNA manipulation, including restriction digestion, ligation,
polymerization, PCR, etc. is done
in vitro
which methodology allows researchers to amplify DBA molecules directly?
polymerase chain reaction - or PCR
PCR (polymerase chain reaction) is used to :
amplify specific DNA
Which region is amplified in PCR:
The region between where two small synthetic DNA primers anneal is amplified
Primers are designed to be :
unique to the region to be amplified and to “face each other”
PCR temperature cycling:
- Denaturation of the DNA - 96C
- Annealing of the primers - 52 degrees celsius
- DNA synthesis from the primer - 72 degrees celsius
Begin Again
thirty cycles of synthesis can create:
10^9 copies of the DNA
PCR requires:
- Template DNA
- Oligonucleotide primers that are specific to the boarders of the region to be amplified
3.DNA polymerase, usually the heat stable TAQ polymerase
*a thermal cycler
PCR cycle:
- Heat denaturation to melt the DNA into single strands (95 degrees celsius)
- Cool the reaction to allow the oligonucleotide primers to anneal to the target DNA (54C)
- Raise the temperature to 72 degrees celsius so Taq polymerase synthesizes a complementary strand of DNA
If you repeat the PCR cycle 30 times, the targeted DNA will be replicated approx:
1 billion times
PCR reactions use:
heat stable DNA polymerases like Taq polymerase, which comes from thermus aquaticus (a bacterium that grows in hot springs)
What is reverse transcriptase?
reverse transcriptase is the enzyme that can copy RNA into DNA (the reverse of the “central dogma”)
reverse transcriptase can be used to :
turn RNA molecules (in particular mRNA molecules), into cloneable DNA molecules, called cDNA for “complimentary DNA”
How can PCR be used to quantify mRNA:
mRNA is used as a template with reverse transcriptase to synthesize a DNA copy of the RNA
DNA copy is called a cDNA (from complimentary RNA)
the first stand of cDNA can be used for:
quantification in a PCR reaction that is referred to as RT-PCR (reverse transcriptase PCR)
The amount of DNA product resulting from amplification by PCR depends on:
how much DNA (or cDNA or mRNA) was in the original smaple
