🧆 🌊 ®️ Flashcards
Polymerase chain reaction (PCR)
amplifies a small amount of DNA into large amounts
What is the name of the sample of DNA needing to be copied
The template DNA (often a known sequence or gene)
Template DNA
Typically double-stranded and only extremely small quantities are sufficient
What are the reagents needed for PCR
- a pair of oligonucleotide primers
- supply of nucleotide triphosphates
- Taq DNA Polymerase (actually makes the copies)
What do the oligonucleotide primers have?
sequences complementary to the ends of the template DNA
Reverse transcriptase PCR (RT-PCR)
utilizes reverse transcriptase to make a cDNA copy of mRNA. PCR is later used to amplify the cDNA. This is useful as eukaryotic DNA tends to have long stretches of noncoding introns.
What are the two steps of RT-PCR?
- reverse transcriptase recognizes the 3’ end of primers containing repeated thymines and creates a DNA strand complementary to the RNA
- The RNA strand is replaced with another DNA strand -> double stranded cDNA that can be amplified.
What ends of the template strand do the primers anneal to?
5’ end of the sense strand and 3’ end of the antisense strand
What is the basic mechanism for PCR?
-denaturation of the template
-annealing of the primers
-making a complementary copy using DNA polymerase
Inverse PCR
Method for using PCR to amplify unknown sequences by circularizing the template molecule
What does the process of PCR require
changing of temperatures (in a cyclic manner)
what is the general sequence of inverse PCR?
DNA is cut with a RE that cuts upstream and downstream but not within it. The linear piece of DNA is circularized and amplified with primers that anneal into the known region. PCR products will have the unknowns that can be cloned.
degenerate primers
primer with several alternative bases at certain positions - they are based on amino acid sequences and contain different nucleotides at the wobble position
What are the modifications between the PCR cycle and DNA synthesis in vitro
-Double strand template is denatured with high heat (not enzymes)
-the temperature is lowered so primers anneal to their binding sites
-primers bind to opposite strands of the template (one at the beginning and one at the end)
What is the most widely used polymerase for PCR?
Taq polymerase (stable and does not denature at high temp.)