Detection of cancer progression markers via PCR III Flashcards
Show how multiple cycles of PCR result in a logarithmic expansion of the DNA
What reagents are required for PCR?
- DNA Polymerase
- Taq DNA Polymerase
- dNTP
- Primers
- MgCl2
- Buffer
Why is DNA polymerase required for PCR?
This is the enzyme that synthesizes DNA molecules from deoxyribouncleotides (the building blocks of DNA).
These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule.
Why is Taq DNA polymerase required for PCR?
It is a thermostable enzyme that synthesizes DNA from single-stranded templates in the presence of dNTPs and a primer.
Why are primers required for PCR?
They areligonucleotides, typically 15-30 bases long, that hybridize to opposite strands and flanking the region of interest in the target DNA
Why is MgCl2 required for PCR?
•Mg2+ ions bind to the catalytic site of the enzyme by working as a cofactor and increases its power to perform the reaction; and the Mg2+ ions of MgCl2 bind to the PO3– of the DNA backbone and protects the negatively charged phosphate of DNA backbone by decreasing electrostatic repulsion between DNA strands.
Which buffer is required for PCR?
50 mM KCl and 10 mM Tris-HCl, pH 8.8
PCR Practical: What sample type will be used?
Colon biopsy samples
PCR Practical: Describe the patient sample preparation
RNA extraction and reverse-transcriptase PCR have been performed to generate a complementary DNA library.
PCR Practical: What 3 samples will be available?
- Positive control
- Negative control
- Patient sample
What is a positve control?
A condition that shows that the experiment is well conceived and will work
What is a negative control used for?
- To determine if the assay is sensitive enough
- To make sure that the problems of contamination will be highlighted when they occur
- To validate the specificity of your primers
Draw a PCR reaction-thermal cyling graph
