MB504T Handouts

HAND OUTS Module # ( 001- 096 )

MODULE# 001 Polymerase chain Reaction

Molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production and health, environment and the industry related to these areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. This technique was developed by Kary Mullis in the mid 80's and since then it has been considered as an essential tool in molecular biology which allows amplification of nucleic acid sequences (DNA and RNA) through repetitive cycles in vitro. The mechanisms involved in this methodology are similar to those occurring in vivo during DNA replication. The amplification of specific nucleic acid sequences, even in the presence of millions of other DNA molecules, is achieved by thermostable DNA polymerase enzyme (as the name of this technique suggests: ―polymerase chain reaction‖) and specific primers. Primers are short sequences of DNA or RNA (oligonucleotides) that initiate DNA synthesis.

Reference:Polymerase Chain Reaction Edited by Dr. Patricia Hernandez-Rodriguez, Chapter # 8: Polymerase Chain Reaction: Types, Utilities and Limitations, Page 157-158.

MODULE# 002 Steps of PCR

PCR is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time.

A typical PCR consists of:

Initial Denaturation: The reaction temperature is increased to 95 °C and the reaction is incubated for 2–5 min (up to 10 min depending on enzyme characteristics and template complexity) to ensure that all complex, double-stranded DNA (dsDNA) molecules are separated into single strands for amplification.

Cycling:

1. Denaturation: The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA).
2. Annealing: In annealing short DNA molecules called primers bind to flanking regions of the target DNA. The temperature is lowered to approximately 5 °C below the melting temperature (Tm) of the primers (often 45–60 °C) to promote primer binding to the template.
3. Extension: The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended.DNA polymerase extends the 3′ end of each primer along the template strands.

Repeat: Steps 1–3 repeated (―cycled‖) 25–35 times to exponentially produce exact copies of the target DNA. Final elongation: Single step is performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold temperature: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

Figure: Three steps of PCR─denaturation, annealing, and extension─as shown in the first cycle, and the exponential amplification of target DNA with repeated cycling.

Reference:

MODULE# 003 Ingredients of PCR

A PCR template for replication can be of any DNA source, such as genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA. Contamination of template with other materials from the sample source can result in PCR inhibition and in extreme cases, complete failure of the assay. DNA polymerases are critical components in PCR, since they synthesize the new complementary strands from the single-stranded DNA templates. All DNA polymerases possess 5′→ 3′ polymerase activity, which is the incorporation of nucleotides to extend primers at their 3′ ends in the 5‘ to 3‘ direction. Oligonucleotide primers are typically 15–25 nucleotides in length with 40–60% GC and a Tm that is similar for each member of the pair. Primer pairs should have a Tm within 5°C of each other. A simple method to assess the Tm of short oligos (<25 bases) is to use the formula: Tm = 4(G + C) + 2(A + T) Deoxynucleoside triphosphates (dNTPs) are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of PCR amplification. dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP.The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the PCR is usually 200 μM. Free Mg2+ ions are required as a co-factor for DNA polymerase activity; however, Mg2+ ions form complexes with dNTPs, primers and DNA templates. Buffer of the PCR reaction mixture serves as a chemical environment to maintain an activity and stability of the DNA polymerase.

Figure: Requirements and purpose of amplification cycles (denaturation, annealing and extension) in a polymerase chain reaction (PCR).

MODULE# 004 Primers for PCR

A primer is a short single-stranded DNA fragment used in certain laboratory techniques, such as the polymerase chain reaction (PCR). In the PCR method, a pair of primers hybridizes with the sample DNA and defines the region that will be amplified, resulting in millions and millions of copies in a very short timeframe. Oligonucleotide primers are typically 15–25 nucleotides in length with 40–60% GC and a Tm that is similar for each member of the pair. A simple method to assess the Tm of short oligos (<25 bases) is to use the formula: Tm = 4(G + C) + 2(A + T). The main property of primers is that they must correspond to sequences on the template molecule (must be complementary to template strand). The size of the primer is very important as well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a long primer is used to amplify a eukaryotic genomic DNA sample. The structure of the primer should be relatively simple and contain no internal secondary structure to avoid internal folding. One also needs to avoid primer-primer annealing which creates primer dimers and disrupts the amplification process.

Primers should generally have the following properties:

Length of 18-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions Figure: Forward and Reverse Primers.

Guo, J., Starr, D., & Guo, H. (2020). Classification and review of free PCR primer design software. Bioinformatics, 36(22-23), 5263-5268.

MODULE# 005 DNA polymerases

The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).The function of Taq polymerase is to replicate the target DNA. The DNA polymerases recognize primers as start tags. The availability of the thermostable DNA polymerase provided the means to automate the reaction. It was originally isolated from Thermophilus aquaticus that thrives in hot springs. Taq DNA polymerase has a temperature optimum of 75–80oC and survives prolonged exposure to temperatures as high as 96oC. So, it can stay active after each of the denaturation steps.

Taq polymerase lacks proofreading activity and unable to correct wrong incorporated nucleotide bases. On average, these errors occur approximately once per 9000 nucleotides. This might be important for some applications, such as cloning, because the product of the polymerase chain reaction may not be a completely precise copy of the original sequence. Another imperfection of Taq polymerase is that it can only efficiently amplify fragments of a few thousand base pairs. Amplification of very long fragments (up to 40 kb) requires special methods. These problems can be solved by using other thermostable polymerases, such as Pfu and Pwo DNA polymerases. These enzymes have proofreading activity.

components-and-their-functions

chain-reaction-pcr#:~:text=Like%20DNA%20replication%20in%20an,was%20isolated%20(Thermus%20aquaticus).

MODULE# 006 Standard PCR reaction

Standard PCR reagents include a set of appropriate primers for the desired target gene or DNA segment to be amplified, DNA polymerase, a buffer for the specific DNA polymerase, deoxynucleotides (dNTPs), DNA template, and sterile water. It also includes a negative control, and a positive control. The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler. 25-30 cycles of amplification are recommended.

Table: PCR reagents in the order they should be added.

Reagent · Concentration · of · stock solutions · Volume · Final Concentration

Sterile H2O Q.S. to 50 μl PCR Buffer 10X 5 μl 1X dNTP's 10 mM 1 μl 200 μM MgCl2 25 mM 3 μl 1.5 mM Forward Primer 1 μl 20 pmol