Hot Start PCR | Multiplex PCR | Avoiding Contamination In PCR | Advantages & Disadvantages Of PCR

Hot Start PCR

• • Amplitaq gold is used in hot-start PCR which is used to encourage specific product formation.

• • To discourage nonspecific product formation by Taq polymerase.

• • 1°C increase in temperature ➡️ 0 02 reductions in pH.

• • Initially starting pH will be 9 It will decrease to 8. Thus, the enzyme will activate. When the temperature rises to the maximum required only then will the enzyme activate.

Multiplex PCR

In multiplex PCR, if less sample is present and more than one reaction has to be carried e.g 17 STR sites have to be amplified then it can be used. For 17 STR sites, as there will be 34 primers and 17 products so there can be chances of diner formation. To avoid this primers should be best to avoid diner formation within a set or other.
There will be high chances of dimer formation in the case of 34 primers so for STR amplification as multiplex PCR  is used has to be optimized first. Primer concentration also has to be optimized i.e. by adding which primers results are better. Primer sequences also have to be optimized. Either kit primers or prepared primers should be used otherwise many reactions have to be carried for optimization.

The concentration of Mg²+ ions and that of DNCTPs have to be increased than normal. 8000 STR sites are reported but those which are used are 13. Different companies use different STR sites.

STR sites can be simple or complex they can be compound . or complex hypervariable. They can be STR or minisatellites. The sequence repeats are 〜16 nt. Repeats can be of di/ tri/ tetra /
Penta / Hexa / or Hepta nucleotides . Tetranucleotides repeats are more in use.

D1S80 ➡️ minisatellite

➡️    10 bp

25 PCR products have been ca amplified repeatedly using a single tube at the same time by using different primer sets.

Real-Time PCR Methods For Quantification

1. Absolute
2. Relative
3. Online Product   Formation
4. More sensitivity 
5. Easy quantification

 

Product Detection 

1. Syber Green
2. Taqman  5΄ nuclease

When synthesis occurs then the probe gets hydrolyzed & quencher separates.
More specificity.
At a time multiple can be amplified by using different probes with different fluorescence different products that can be detected.

Avoiding Contamination In PCR

1. Work in laminar flow
2. Use sterile PCR tubes & micropipettes
3. Rnase-Dnase free buffer
4. Single-use of each tip
5. Proper storage of reagent
6. Frequent change of gloves
7. Wash hands with ethanol
8. Pre PCR, Past PCR & PCR should be physically separated. (Experimental and reference sample be in separate labs)
9. Aerosol resistant tips
10. UV irradiation
11. Clean the apparatus with 10% bleach & isopropanol.

Advantages Of PCR

1. Specifically amplifies template DNA
2. Small amounts of template DNA are required.
3. Can deal with degraded DNA
4. No contamination with fungal or bacterial DNA and even if so then they cannot be amplified as primers are specific.
5. Can go for multiplex
6. Commercial kits are available

Disadvantages Of PCR

1. There can be some inhibitors.
2. There can be contamination from other humans i.e. lab workers etc.
3. There can be chances that for a specific STR there will be no product. There may be mutations in that region where primers were to bind due to which they will not bind hence no product formation.

Editor's Recommendation:

• Analysing Metabolic Pathways
• Protein Threading Sequence
• Ab Initio Protein Structure Prediction
• Homology Modeling
• DNA Damage
• Docking | Protein-Protein Docking | Protein-Ligand Docking 
• Functional Regulation | Genetic Aspect | Indirect Aspects 
• Database Development 
• Functional Analysis At Structure Level
• PTMs and Functional Regulations
• Immuno Quantitative Assay 
• Modeling Cellular Processes
• PCR Reagents | Stochastic Effect | STR Classification
• DNA Degradation
• DNA Quantification | Human DNA Quantification Method | Advantages  
• Desirable Characteristics of STR used in Forensic DNA typing
• DNA Ladders
• Metabolic Pathways
• Non-Human DNA
• Mitochondrial DNA 
• Integrated Genomic Circuits 
• Shutter Product Formation 
• STR Sites
• Mini STR Sites 
• Molecular Diagnosis of Genetic Diseases
• Real-Time PCR