PCR 2- Neil Flashcards
(32 cards)
Key principles of PCR:
Process of DNA replication
Requiring only a few of the biological components (proteins and chemicals)
Mechanical/electronic device
Heat source to denature DNA into single strands (no need for other proteins)
Facilitates exponential artificial DNA amplification
Used in a variety of Clinical, Commercial and Forensic diagnostic applications
PCR is a technique which specifically amplifies a piece of DNA
Q. How do we obtain the gene that we would like to amplify?
A) By using the Method called Bioinformatics (Biological informational analysis) through Pubmed
B) Selecting the gene that we wish to look at/amplify
PCR
Manufacture DNA Primers (commercially available) 20 bases in length
Purchase some DNA polymerase
Availability of a PCR Machine
Begin the assay
What can be achieved through PCR?
Through such repetitive cycles it is possible to obtain millions/billions of copies of the desired DNA segment within a few hours. The procedure is very simple, requiring (in theory) only a test tube and some heat sources (same source or three different sources.
If the reaction starts with 1 copy of a piece of DNA then for each cycle the reaction will amplify double the amount…
End of Cycle 1 = 2 copies
End of Cycle 2 = 4 copies
End of Cycle 3 = 8 copies
End of cycle 4 = 16 copies
End of cycle 5 = 32 (25) copies
End of cycle 32 = 2(exp 32) copies
= 4 294 967 296
Q, If A reaction starts with 15 copies
What would be the number of copies after 33 cycles?
A. 15 X 2 (exp^33) = 128 849 018 880
Qualitative PCR
A standard protocol to detect the presence (Quality) of a single DNA molecule in a sample solution
Qualitative PCR further information:
When PCR is used only for detecting a specific DNA segment, the method is referred to as qualitative PCR
Medical applications of the classical PCR method is therefore the detection of pathogens (eg Detection of HIV, HBV)
PCR is able to detect the DNA or RNA of the pathogens much more quickly
PCR method can detect the DNA of micro-organisms in any sample, whether of body fluids, foodstuffs or drinking water
PCR is helping to solve is to determine if donated blood is contaminated (HIV or Hep C)
Has been used to determine whether solutions have been tampered with (Oil, Counterfeit goods)
What can qualitative PCR detect?
- Qualitative detection of Hepatitis (B) Viral particle (HBV) DNA in serum.
- DNA-based Steganography
What is DNA-based Steganography?
Steganography is the practice of concealing a file, message, image, or video within another file, message, image, or video.
Patented PCR Method of Identification and Authentication
The invention is a stenographic method for concealing coded messages in DNA. The method involves concealing a DNA encoded message within a genomic DNA sample followed by further concealment of the DNA sample to a microdot. The present invention further provides a method for the use of genomic steganography to mark and authenticate objects of interest
- DNA based approach to confirm/deny bacterial contamination of commercial products
The PCR food-proof Beer screening Kit enables the detection of contaminating bacteria in a single PCR test with high sensitivity (down to 101 cfu/ml)
Furthermore, the LightCycler food-proof Beer screening Kit allows the specific identification of individual beer spoilage bacteria using melting-curve analysis.
Other Food based uses for Qualitative PCR DNA analysis:
- Horse meat contamination of Beef products (discriminate between meat sources)
- DNA in foodstuffs derived from or containing genetically modified organisms (GMO)
- Authentication of Plant Oils & Food traceability
- Quantitative molecular biology analysis of complex food matrices for the detection of food fraud (e.g. Rice, Fish)
- Qualitative polymerase chain reaction methods for detecting major food allergens (peanut, soybean, hazelnut, peanut, charlock, celery, sesame, lupine,
walnut, almond, macadamia nut, hickory and wheat)
- DNA Based marking system:
TraceTag (Cardiff) provides a secure system for the marking of branded oil products to prove brand identity and the presence of specific brand additives and their concentration to ensure customer confidence. TraceTag adds a small amount of DNA Tag to the crude Oil at source to help determine a variety of parameters (others Seeker DNA, Selecta DNA)
Smuggling of fuel is a major problem in many parts of the world with the varying tax regimes in neighbouring countries. PCR can be used to;
1.Determine the origin of the fuel.
2. Determine the theft of oil (proof of innocence or guilt)
3. Identification of Sources of pollution (Ecological)
- Criminal Identification, SelectaDNA products:
SelectaDNA Gun
containing pellets with unique artificial genetic sequences contained within the pellet gel.
SelectaDNA Gel
Uniquely identify criminals tampering with indoor property or illegally gaining access to premises.
SelectaDNA Defence DNA Spray
Spraying DNA molecules with UV particles to help police in identifying the perpetrators of crime.
Detection:
Agarose gel and/or Polyacrylamide gel electrophoresis Restriction endonuclease digestion
Dot blots
High-pressure liquid chromatography
Electrochemiluminescence
Direct sequencing
Visualisation:
-EtBr staining (UV transilluminator,
image analyzer)-Southern blotting (hybridization with labeled probe)
-Incorporation of label into amplifying sequence
-Silver staining
-Agarose or polyacrylamide gel-HPLC
-Hybridization with labeled probe
UV detection
Voltage-initiated chemical reaction/photon detection
Radioactive or fluoescent-based DNA sequencing
The product of PCR:
The product of a PCR reaction should be a fragment or fragments of DNA of defined length. Many techniques can be used to detect amplified sequences .
The simplest and commonly used technique is electrophoresis of the PCR product on an Agarose gel with a DNA stain EtBr (ethidium bromide).
Detection of PCR products:
- Discontinuous (end point analysis - finished PCR amplification)
- Gel Electrophoresis (GE)
- Agarose (AGE)
- Polyacrylamide (PAGE)
- Continuous (after each amplification cycle)
(Real Time or; Quantitative PCR or Q- PCR)
Other PCR based tests:
PCR ELISA’s (amplified material detected via elisa vs Biotin)
Real Time PCR (PCR amplicon measured after every cycle)
PCR-PNA-HPLC (Peptide nucleic acid combined with high performance liquid chromatography)
Duplex PCR (2 PCR products amplified at same time)
Multiplex PCR (several different genes amplified simultaneously)
Quantitative PCR:
PCR technique is used to calculate the amount (quantity) of a particular DNA sequence within a biological specimen
-Blood
-Tissue
-Water
-Urine
Quantitative PCR continued:
Measure the quantity of the DNA/RNA in any sample
Applicable to measuring the amount of mRNA (reflection of gene expression) using Reverse Transcription PCR (RT-PCR)
Exercise (switches genes on/off)
Hormones (switches genes on/off)
Prokaryotic Food Sources (switches genes on/off)
The Quantity of the starting material dictates the kinetics of DNA amplification
Principle of quantitative PCR:
In order to measure mRNA you first have to convert the mRNA to DNA
DNA polymerase cannot use RNA as a template (needs DNA)
Requires the use of an additional enzyme which converts mRNA to DNA (derived from a virus)
Called Reverse Transcriptase
Enzyme helps to measure
GENE EXPRESSION = Quantitative mRNA
