Molecular Diagnostics Nucleic Acid Amplification by PCR Flashcards
Who invented PCR?
Kary Mullis
Terminology:
-Produces many copies of DNA
-Most utilized molecular technique
Polymerase Chain Reaction (PCR)
Terminology:
One copy of target DNA can yield billions of copies
Amplification
Applications of PCR (3)
-Infectious disease testing
-Genetic defects
-Forensics
Know A-E
A: Taq DNA polymerase
B: Primers
C: dNTPs (deoxynucleoside triphosphate)
D: Buffer
E: DNA
Three steps of standard PCR
- Denaturation
- Annealing
- Elongation/Extension
Temperature range for denaturation
94-96C
PCR Steps:
-Breaks hydrogen bonds
-Separates target DNA strands
Denaturation
Temperature range for annealing
50-65C
Elongation/Extension occurs at what temperature?
72C
Why does elongation take place at 72C?
It’s the optimal temperature for Taq polymerase to efficiently build new DNA strands
PCR Steps:
Primers hybridize to denatured DNA strands
Annealing
PCR Steps:
Taq polymerase synthesizes new complementary DNA strands
Elongation/Extension
Why does Elongation/Extraction occurs at 72C?
It’s the optimal temperature for Taq polymerase to function
Components for PCR (6)
-Sample
-Primers
-Nucleotides
-Taq polymerase
-Mix buffer
-PCR tube
What is Taq polymerase?
A DNA polymerase commonly used in PCR
Where does Taq polymerase come from?
A heat-tolerant bacterium known as Thermus aquaticus
In vivo, what is responsible for denaturation of DNA? (enzyme)
Helicase
In vitro, how is DNA denatured?
Heat
Why do G-C pairs requires more energy (or heat) for the primer to bond to the target strand when compare to A-T?
G-C pairs have 3 hydrogen bonds whereas A-T pairs only have 2 hydrogen bonds
Terminology:
Products of PCR (all the copies of DNA)
Amplicons
What are 3 post-PCR analysis methods?
-Gel electrophoresis
-Labeled probes
-DNA sequencing
TRUE or FALSE:
Standard PCR is limited to length limitations of up to 1000 base pairs
TRUE
3 ways to prevent contamination for standard PCR
-Creation of aerosols
-Physical separation of space if possible
-Unidirectional workflow
PCR Variations (4)
-Multiplex PCR
-Reverse transcriptase PCR
-Real Time PCR
-Other PCR
2 types of “other PCR” mentioned
-Nested PCR
-Sequence-specific PCR
Which PCR variation:
-mRNA used to synthesize cDNA
-Testing for RNA viruses
-RNA is fragile
RT-PCR
Which PCR variation:
-TaqMan probe
-Uses FRET
-Sequence-specific
-Allows for quantization of starting material in samples
-Expensive
qPCR
2 main types of detector in qPCR
-Non-sequence specific dyes (SYBR Green)
-Sequence-specific probes (TaqMan and Molecular Beacon)
Which PCR variation:
-Simultaneous detection of multiple targets in a single tube/well
-SNP genotyping, pathogen detection, forensics
-More information with smaller sample size
-Primer design can be complex
Multiplex PCR
What is Multiplex PCR commonly used for?
Panel testing
What is Reverse Transcriptase PCR commonly used for?
-Testing RNA viruses
-Detecting gene expression in cancer cells
Which qPCR detector uses fluorescence energy resonance technology (FRET)?
Sequence-specific probes:
-TaqMan Probes
TaqMan probes used in qPCR utilize FRET, which stands for what?
Fluorescence Resonance Energy Technology
On a qPCR amplification plot, what does the Ct value represent?
Cycle threshold
