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Purposes of Nucleic Acid extraction
To separate nucleic acid containing cells of interest from their environment and then separate DNA or RNA from those cells and their components
To obtain useful samples of DNA or RNA that are free from contaminants that could impede testing
To isolate DNA or RNA of sufficient quantity and quality to perform analysis
Specimen types for nucleic acid extraction
Whole blood (EDTA)
Bone Marrow
Solid Tissue
Serum/plasma/cell-free DNA
Amniocytes
Products of conception
Buccal swaps
Saliva, sputum
Endocervical swabs
Urine, feces
Pus
Basic steps in nucleic acid extraction
Pretreatment of cells, if necessary
Lysis of WBCs or other nucleated cells
Denaturation/digestion proteins
Purification of DNA/RNA
Resuspension in buffer
What happens in pre-treatment?
Blood and bone marrow - remove RBCs
Digest paraffin wax if necessary
Tissue - grind or mince
Microorganisms - digest cell walls
What happens in Lysis
Detergent - SDS
Liberates contents of the cells: proteins and nucleic acid
Separates histone proteins from DNA
What happens during digest proteins and nulceases?
Enzymatic degradation by incubation with an enzyme - protease/proteinase
Also targets nucleases
DNase
RNase
Can add reagent RNase to degrade RNA
What happens during purification?
Protein breakdown products are separated from free DNA/RNA
AKA protein precipitation
Can be done using a solvent (liquid phase extraction) or a filter column (solid phase extraction)
What happens during Nucleic Acid precipitation
alcohol use to concentrate DNA/RNA
Usually cold ethanol or isopropanol
DNA and RNA insoluble in alcohol
suspension centrifuged, alcohol discarded
What happens during resuspension
Concentrated DNA/RNA pellet resuspended in buffer or nuclease free water
Concentration measured and adjusted prior to testing
Purity also assessed
Nucleic acid quantitation
Allows the determination of the efficiency of a purification technique
Different assays require different concentrations of nucleic acid
Spectrophotometry
Spectrophotometry for QUANTITY
Measurement of how much a substance absorbs or transmits light
Absorbance used to calculated concentration
Nucleic acids absorb light at 260 nm, via adenine
Absorptivity constants:
50 μg/mL for DNA
40 μg/mL for RNA
Spectrophotometry for QUALITY
Proteins absorb light at 280 nm
Measure extracted nucleic acid at both 260 nm and 280 nm and calculate ratio of two reabsorbance values
Pure DNA should have a ratio between 1.6 and 2
Pure RNA should have a ratio between 2 and 2.3
Can reprecipitate or further purify if necessary
How does conventional PCR compare to replication
In vitro:
Target DNA duplex is denatured by raising temperature to 95 degrees C
Primer bound to denatured strands of target DNA
First round of amplification
Second round of amplification - the newly synthesized strand terminates at the opposite primer site
Third round of amplification - both strands are truncated (shortened) at the primer sites
In Vivo:
The hydrogen bonds between the two strands of DNA are broken by the enzyme DNA helicase
Replication begins at short RNA primers that bind to DNA. The RNA primers are later removed and replaced with DNA
Typically occurs at 37 degrees C (Body temperature)
Makes a single copy of all of the DNA in the nucleus
Three main steps in a cycle
Denaturation - 95 deg C
Annealing - 55 deg C
Elongation - 72 deg C
Exponential amplification
After each cycle of PCR the amount of strands multiplies by 2
Components and functions of reaction mix
Primers - Helps to limit DNA that is copied by binding to beginning and end of region to be copied
Taq DNA polymerase - nucleotides / dNTPs complimentary to the template strand
MgCl2 + buffer - Helps Taq polymerase function properly
dNTPs - nucleotides (A,T,C,G) used by Taq to build new DNA
Controls and their purposes
Blank reaction
All reagents without DNA
Ensures reagent mix not contaminated
Negative control reaction
All reagents, with DNA known to lack target
Ensures primer specificity
Positive control reaction
All reagents, with DNA known to contain target
Ensures all aspects of assay working properly
Amplification/ internal control reaction
All reagents, with second primer set, unrelated to target but present in all samples
Ensures DNA sample does not contain inhibitors
Distinguished between true negative and false negative
Why do we use Reverse Transcriptase (RT) PCR
Essential for analysis of gene expression (RNA transcript levels)
How does RT-PCR work?
PCR is preceded by synthesis of DNA from RNA
Utilizes reverse transcriptase from retroviruses
ssRNA reverse transcribed into complimentary DNA (cDNA) - RNA hybrid
cDNA is then used as template for PCR, to amplify gene of interest
amount of cDNA template is proportional to starting RNA amount/level of gene expression
Unlike genomic DNA, cDNA does not contain “junk DNA“ /introns
Major steps in RT-PCR
RNA extracted from specimen
RNA + non-specific, random primers incubated briefly at high temp
Denatured RNA added to master mix containing
Reverse transcriptase
dNTPS, Buffer, RNase inhibitors
Primers extended at ~42 deg C for an hour (1 cycle)
Reaction mix heated briefly to denature RT enzyme (~95 deg C)
PCR reaction performed to amplify target DNA, if present