PCR and Spectrophotometry Flashcards
PCR
DNA or RNA sequences can be amplified
Developed by Kary Mullis - 93 Nobel Prize
Fast, robust, very simple
Different types and applications, such as diseases, diagnosis, forensics, research
Exponential amplification
PCR and amplification
Selectively amplifies DNA sequences, increases the copy number
An exponential amplification
Stops at end of reagents
6 PCR Components
- Template
- Buffers - provide suitable enviro
- Mg2+ - needed to DNA polymerase will bind dNTPs
- dNTPs - the building blocks
- Primers
- DNA Polymerase
Template
Contains the target region to be amplified: plasmid, genomic DNA, cDNA
Buffers
Provide a suitable chemical environment for optimum activity and stability of the DNA polymerase
-usually have magnesium as buffer, rxn will not happen without
Mg2+
Usually as magnesium chloride needed so that DNA polymerase will bind dNTPs
Deoxynucleoside triphosphate
dNTPs
the building blocks from which the DNA polymerases synthesizes a new DNA strand
Primers
Complementary to the DNA regions of the 5’ or 3’ ends of the DNA region
DNA Polymerase
Thermostable enzymes (stable at 95’C), eliminating the need to add fresh enzyme after each denaturation step.
- Taq no proofreading ability, induces error, most common
- Pfu has proofreading ability, for cloning purpose
PCR Controls
NTC - no template control - make sure not contaminated
Positive control - what you know works
3 Steps to PCR Reaction
- Denaturation - breaks H bonds to dissociate
1 minute at 94 C - Annealing - forward and reverse primers
45 seconds at 56-62 C - Extension - only dNTPs
2 minutes at 72 C
5 Rules to Primer Design
- Length 18-22 nucleotides
- Duplex stability - similar melting temps
- No hairpin turns
- Non-complementary so don’t go with each other
- Optimal distance 150-500 bp
Extension Temperature
Depends on DNA polymerase that that is utulized. This is the are of the PCR cycle that nucleotides are added to the end of the primer strands and a new strand of DNA is synthesized
- 72 C, 1 min/1 kb of DNA to be amplified when using Taq DNA polymerase
ex. 7000 = 7 minutes. The longer the extension, the more likely for error
PCR data analysis
Analyzed by agarose and polyacrylamide gel electrophoresis
A molecular weight marker is utilized to identified the size of the amplicons
Smaller migrates faster
4 PCR Types
Nested - PCR within PCR
Multiplex - look at multiple things, common in clinical
qRT-PCR - quantitative real time PCR. used for gene analysis expression
RACE - not a lot of info about sequence, use this
PCR Applications in Medicine
Presence or absence of mutations
Transfusion - blood typing
Tissue typing - organ transplant
Cancer detection - Breast CA - BRCA 1 and BRCA 2
Characterization and detection of infectious disease
Other PCR Applications
Detection of contaminants - air, foot, water
Forensic genotyping - Identify trace human tissue
Research - gene expression, single-nucleotide polymorphism (SNP) detection - personalized medicine
Spectroscopes
Measure electromagnetic emission
Spectrophotometers
Measure electromagnetic absorption
Spectrophotometry
Process that determines the amount of light absorbed by colored compounds
UV
Ultraviolet
200-300nm
VIS
Visible
400-800nm
blue-green-yellow-orange-red
A260 Unit
Used as the quantity measure for nucleic acids. Depending on nucleic acid, factor will be:
A260 ds DNA = 50 ug/ml
ss DNA = 37 ug/ml
ss RNA = 40 ug/ml
Wavelength for detecting DNA
is 260nm
Wavelength for detecting protein
is 280nm
Formula to calculate the concentration is
Concentration = A260 x dilution x nucleic acid factor
Pure DNA A260/280
1.8
Pure RNA A260/280
2.0
Colorimetry
Solutions of many compounds have characteristic colors
Intensity is proportional to the concentration of the compound
Bradford, bicinchoninic acid assay (BCA), Lowry assays
