Molecular Exam 2

Purposes of Nucleic Acid extraction

1. To separate nucleic acid containing cells of interest from their environment and then separate DNA or RNA from those cells and their components

2. To obtain useful samples of DNA or RNA that are free from contaminants that could impede testing

3. 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

1. Pretreatment of cells, if necessary

2. Lysis of WBCs or other nucleated cells

3. Denaturation/digestion proteins

4. Purification of DNA/RNA

5. 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

1. Denaturation - 95 deg C

2. Annealing - 55 deg C

3. Elongation - 72 deg C

Exponential amplification

After each cycle of PCR the amount of strands multiplies by 2

Components and functions of reaction mix

1. Primers - Helps to limit DNA that is copied by binding to beginning and end of region to be copied

2. Taq DNA polymerase - nucleotides / dNTPs complimentary to the template strand

3. MgCl2 + buffer - Helps Taq polymerase function properly

4. 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

1. RNA extracted from specimen

2. RNA + non-specific, random primers incubated briefly at high temp

3. Denatured RNA added to master mix containing

   1. Reverse transcriptase

   2. dNTPS, Buffer, RNase inhibitors

4. Primers extended at ~42 deg C for an hour (1 cycle)

5. Reaction mix heated briefly to denature RT enzyme (~95 deg C)

6. PCR reaction performed to amplify target DNA, if present