DNA extraction and PCR Flashcards
Portion of genome encoding proteins?
1.2% of genome
Function of miRNA in clinical biochemistry?
An interesting biomarker - diagnostic e.g. cancers
How is the nucleic acid prepared?
Nucleic acid isolation, amplificaiton, detection analysis or quantification
What is the stepwise action of nucleic acid purification?
Release nucleic acids by lysing the cell/organism
Seperate nucleic acid from other cell or sample material, including protein
Purify NA by washing away unwanted material
Concentrate (optional - dependent on next step). Any time in the process, increase the concentration of target organism or NA
What sample types are appropriate for NA purification?
Blood collected in an anti-coagulant, WBC used (heparin will interfere with PCR if not completely removed)
Tissue samples e.g. cancer
Pathogens
Lysis of the cell can be …..
A. Mechanical
B. Chemical
C. Enzymatic
All can release NA from cells
Often simple, especially for human cells, e.g. detergent
How is protein removed?
Proteins like histones surrounding NA need to be removed.
Nucleases need to be removed, because it can break down the NA.
Chemical and enzymatic techniques can degrade or precipitate proteins e.g. SDS
Addition of proteases can remove unwanted proteins
How is isolation of NA undertaken?
Liquid liquid extraction or Solid phase extraction
Describe liquid liquid extraction with an example.
Different solubility in immiscible liquids e.g. phenol denatures proteins whilst nucleic acid stays in aqueous phase, which can be seperated using chloroform and isoamyl alcohol
- NA is then precipitated in the presence of alcohol and high conc of salt, then centrifuged into a pellet
Describe solid phase extraction with an example.
Techniques include - ion exchange chromatography (using negative charge of DNA)/affinity chromatography
More common and less hazardous - also easier to automate
Analytical methods to measure quality and quantity of NA?
UV absorbance or fluorescent staining
Describe UV absorbance of NA.
NA absorbs UV light at 260nm, but it cannot distinguish between DNA and RNA.
Reference pure dsDNA at 50mg/L has an abs of 1 at 260nm
How is purity of NA estimated using UV absorbance?
Using a ratio of absorbance at 260nm and 280nm (280 is proteins)
Pure preperation should have A260/A280 of 1.7 to 2
What are some advantages of fluorescent staining?
- higher sensititivity and no background binding (interference), e.g. ethidium bromide in gel electrophoresis
- Real time PCR uses dyes in solution
Once purified what is NA used for?
Polymerase chain reaction (PCR)
- method to amplify NA
- denaturation of target DNA
- annealing of specifc primers
- extension of primers using a thermostable DNA polymerase enzyme (Taq DNA polymerase)
Common uses for PCR
When there is insufficient NA in a sample for reliable detection, e.g. dot-blot
If other methods of aetiological diagnosis are unsuitable - direct isolation of organism e.g. SARS-CoV-2
When a rapid result is desired
If a large amount of the NA is required for analytical purposes
Describe the basics of PCR and what each stage does.
Strand seperation
- Heat dsDNA to 95C for 15s to melt and seperate strands, denaturing H-bonds
Hybridisation of primer
- Cool to 50-65C to allow primers to anneal to the DNA strands
DNA synthesis
- Heat to 72C to allow elongation. This extension is usually performed by free nucleotides with taq polymerase
How many times are the PCR cycle repeated?
20-30 times - with exponential increases per cycle
Components of PCR.
Target DNA, Primers, deoxynucleotides (dNTPs), Taq DNA polymerase, Buffer
What is the first step when using a single stranded NA?
Reverse transcription to produce a ds
- a cDNA copy is synthesised via incubation of the target sequence with reverse transcriptase, an appropriate primer and dNTPs
What are some key features of primers?
- 15-30 bases
- No base repetitions more than 3-4
- complementary: dont want it to fall on itself
- Possibility of secondary structures, e.g. hairpin loop
- Balance of bases
How is annealing optimised?
By designing primers with similar melting temp Tm. Annealing is normally 5C below Tm
What are some reaction components of PCR?
- Primers: need to be in excess (95% unused)
- dNTPs: can be modified e.g. labelled
- Taq DNA polymerase: 72-75C (thermostable so can stay in system the whole time), read 5´-3´, has 5´ exonuclease activity
What is the purpose of a buffer in PCR?
To provide a suitable chemcial environment for activity of Taq DNA polymerase
Name examples of buffers available.
- Tris-HCl
- MgCl2
- KCl
- Gelatin
- Denaturing agents : alters Tm
Why is contamination a big problem in PCR?
PCR can detect a single copy of the target sequence - thus a high risk of FP
Scrupulous cleaning of labs and equipment essential
Isolation of pre and post PCR procedures
How can we test for contamination in PCR?
Using negative controls, which can detect during extraction and during PCR.
How is the quality of the process controlled?
Using a positive control (group with receiving treatment with known results)
What are some other types of PCR?
- RT-PCR
- Hot start PCR
- Nested PCR
- Multiplex PCR
- Touchdown PCR
What is RT-PCR?
(reverse transcriptase PCR)
- For single stranded NA
- produce cDNA from target sequence using reverse transcriptase
- Then normal PCR cycles
What is hot start PCR?
Hot start PCR (reduce non-specific priming)
- warm all reagents to 94C before addition of Taq polymerase
- Use heat-activated Taq-polymerase
Hot Start PCR significantly reduces nonspecific priming, the formation of primer dimers and often increases product yields
What is nested PCR?
- 2 rounds of amplification
- 1st round produces large amplicon
- 2nd round with new primers amplify fragment within 1st round amplicon
Intended to reduce non-specific binding in products due to the amplification of primer binding sites
Increase the selectivity of the PCR product
What is multiplex PCR?
- amplification of several sequences in one
- reaction using multiple primer pairs
- useful for single-step differential diagnosis
What is touchdown PCR?
Change in annealing temp by 1C every cycle for the first 10 cycles.
- enhances specificity
- avoid amplifying nonspecific sequences
- increase specificity of the reaction at higher temp and increases the efficiency towaeds the end by lowering the annealing temp
What are some PCR detection methods?
Electrophoresis
- agarose
- polyacrylamide
Defines the size of any amplified products, but does not confirm identity
Confirmation via hybridisation
- Southern blot
- Dot blot
- HPLC
- ELISA
What is real-time PCR? and how is it relevant to a clinical setting?
Used in COVID PCRs
Rapid cycling of small volumes - 1 cycle 30-60s
Rapid detection of fluorescent dye during cycling
- no need for electrophoresis
- confirmation with specific probe
Relative/absolute quantitation
- standard curve
- compare with known sample
Is it more advantageous to use real time PCR compare to traditional?
Traditional PCR is measured at end point, while real time collects data in the exponential growth phase
An increase in reporter fluorescent signal directly proportional to the number of amplicons generated
- increased dynamic range of detection
- No-post PCR processing
- Can detect even a 2-fold change
What is threshold line?
Level of detection
The point at which a reaction reaches a fluorescent intensity above background
What is cycle threshold, CT?
The cycle at which level of detection is reached
Example of real time assay fluorescence.
SYBR green dye
TAQMAN
- exploits 5´ nuclease activity of taq polymerase
- its an oligonucleotide probe with fluorescent reporter at 5´ end and quencher at 3´ end
- Anneals downstream from primer site if target is present
What is in situ hybridisation and its relevence to PCR?
- Allows for precise localisation of specific segment of NA within histologic section
- Underlying basis of in situ hybridisation
- NA can be detected through application of a complementary strand of NA to which a reporter molecule is attached
Application of ISH
- Comparison of presence of specific target with pathology
- Determination of structures and cell types affected
- Developmental biology (gene expression profiling in embryonic tissues)
- Karyotyping and phylogenic analysis
Limitations of ISH
- Fragility of tissue of sections
- damage to tissue during processing
- specificity of probe
- endogenous reactants
What is restriction endonuclease analysis?
Restriuction endonuclease recognise a particular sequence in DNA and cut at specific sites where appropriate base sequence occurs
- Protect bacteria against viral invasion, e.g. EcoRI recognises 5´GAATTC
Describe restriction fragment length polymorphism.
Examines size variation of restriction fragments - Illustrates genotypic differences
- E.g. restriction enzyme activity modified by single nucleotide polymorphisms
May determine resistance mechanisms. Associated with pathogenesis
How is restriction fragment length polymorphism results examined?
Using electrophoresis.
- In normal the fragment is broken into two bands, but mutant show as a singular band
What are microarrays?
The hybridisation of NA to a very large set of oligonucleotide probes - probes attached to a solid support
Application of microarrays
Gene expression analysis and genetic variation analysis
Green and red dyes used.
Yellow= overlap between dyes and that gene was neither strongly expressed nor in cancer cells
