PCR- POLYMERASE CHAIN REACTION AND ITS TYPES

 PCR- POLYMERASE CHAIN REACTION AND ITS TYPES

Types of PCR

                                            i.            Multiplex PCR

                                          ii.            Nested PCR

                                        iii.            q-PCR

                                        iv.            Immune capture PCR

Multiplex-PCR

Multiplex PCR (polymerase chain reaction) is a technique used to amplify multiple target DNA sequences simultaneously in a single reaction. It is a cost-effective and efficient method for analyzing multiple genes or DNA regions in a single sample. Here is a general procedure for performing a multiplex PCR 

Materials:

1)      PCR machine

2)      PCR tubes/strips

3)      Pipettes and tips

4)      PCR primer sets (oligonucleotides) specific for each target sequence

5)      Taq polymerase enzyme

6)      dNTPs (deoxynucleotide triphosphates)

7)      DNA template

8)      Buffer solution (provided with Taq polymerase)

Procedure:

1. Design specific primer sets for each target sequence. These primers should be of similar length and have similar annealing temperatures.

2. Prepare a reaction mix by combining Taq polymerase enzyme, dNTPs, buffer solution, and each primer set in separate tubes.

3. Add the DNA template to each reaction mix.

4. Label each reaction tube according to the primer set used.

5. Load the tubes into the PCR machine and run the program according to the specific parameters for your PCR machine.

6. After amplification, analyze the products by running the PCR products on an agarose gel. The size of the products should match the expected product size for each target sequence.

It is important to optimize the reaction conditions for each primer set to ensure successful amplification of all target sequences. This may include adjusting the primer concentrations, annealing temperatures, and MgCl2 concentration. Additionally, it is important to use appropriate controls to confirm the specificity of the PCR products, such as a negative control without DNA template and positive controls with known target DNA.

Nested-PCR

Nested PCR is a technique used to increase the specificity and sensitivity of PCR amplification. It involves two rounds of PCR, where the second PCR is performed using a small amount of the product from the first PCR as a template. Here is a general procedure for performing a nested PCR

Materials:

1)      PCR machine

2)      PCR tubes/strips

3)      Pipettes and tips

4)      PCR primer sets (oligonucleotides) specific for each target sequence

5)      Taq polymerase enzyme

6)      dNTPs (deoxynucleotide triphosphates)

7)      DNA template

8)      Buffer solution (provided with Taq polymerase)

Procedure:

1. Design two sets of specific primer pairs for each target sequence. The first set of primers should anneal to the outside regions of the target sequence, and the second set of primers should anneal to the internal regions of the first PCR products.

2. Prepare a reaction mix for the first PCR by combining Taq polymerase enzyme, dNTPs, buffer solution, and the first set of primers.

3. Add the DNA template to the first PCR reaction mix.

4. Run the first PCR according to the specific parameters for your PCR machine.

5. Transfer a small amount (e.g., 1-5 μL) of the first PCR product to a new tube for the second PCR.

6. Prepare a reaction mix for the second PCR by combining Taq polymerase enzyme, dNTPs, buffer solution, and the second set of primers.

7. Add the first PCR product to the second PCR reaction mix.

8. Run the second PCR according to the specific parameters for your PCR machine.

9. Analyze the products by running the PCR products on an agarose gel. The size of the products should match the expected product size for the internal region of the target sequence.

It is important to optimize the reaction conditions for each primer set and PCR cycle to ensure successful amplification of the target sequence. Additionally, it is important to use appropriate controls to confirm the specificity of the PCR products, such as a negative control without DNA template and positive controls with known target DNA.

qPCR (quantitative PCR)

qPCR (quantitative PCR), also known as real-time PCR, is a technique used to measure the amount of a specific DNA sequence present in a sample. It uses fluorescent probes or dyes to monitor the amplification of the target sequence during the PCR reaction. Here is a general procedure for performing qPCR

Materials

1)      qPCR machine

2)      qPCR tubes/plates

3)      Pipettes and tips

4)      qPCR primer/probe set specific for the target sequence

5)      Taq polymerase enzyme

6)      dNTPs (deoxynucleotide triphosphates)

7)      DNA template

8)      Buffer solution (provided with Taq polymerase)

- Fluorescent dye or probe specific for qPCR analysis (e.g., SYBR Green or TaqMan)

Procedure:

1. Design a specific primer/probe set for the target sequence. The probe should be labeled with a fluorescent dye and a quencher.

2. Prepare a reaction mix by combining Taq polymerase enzyme, dNTPs, buffer solution, the primer/probe set, and the fluorescent dye or probe specific for qPCR analysis.

3. Add the DNA template to the reaction mix.

4. Load the reaction mix into qPCR tubes/plates, making sure to include appropriate controls such as negative controls without DNA template and positive controls with known target DNA.

5. Load the qPCR tubes/plates into the qPCR machine and run the program according to the specific parameters for your qPCR machine.

6. Monitor the fluorescence intensity during the PCR reaction. The amount of fluorescence generated is proportional to the amount of the target sequence present in the sample.

7. Analyze the qPCR data using appropriate software to determine the threshold cycle (Ct) value, which is the cycle number at which the fluorescence intensity crosses a predefined threshold.

8. Calculate the amount of target DNA present in the sample using a standard curve generated from a series of known concentrations of the target DNA.

It is important to optimize the reaction conditions for each primer/probe set and qPCR machine to ensure accurate and reproducible results. Additionally, it is important to use appropriate controls and statistical analysis to confirm the specificity and sensitivity of the qPCR data.

Immune capture PCR (IC-PCR)

Immune capture PCR (IC-PCR) is a technique used to detect and amplify low levels of target DNA or RNA in a sample using immunomagnetic beads. This technique involves the capture of the target DNA or RNA by specific antibodies that are immobilized on magnetic beads, followed by PCR amplification of the captured target sequence. Here is a general procedure for performing immune capture PCR:

Materials:

1)      Magnetic beads coated with specific antibodies for target DNA/RNA

2)      PCR machine

3)      PCR tubes/strips

4)      Pipettes and tips

5)      Taq polymerase enzyme

6)      dNTPs (deoxynucleotide triphosphates)

7)      DNA/RNA template

8)      Buffer solution (provided with Taq polymerase)

9)      PCR primer sets specific for the target sequence

Procedure:

1. Prepare the magnetic beads by incubating them with specific antibodies for the target DNA or RNA. This will immobilize the antibodies on the surface of the beads.

2. Add the sample containing the target DNA or RNA to the magnetic beads and incubate for a specific time period. The antibodies on the beads will bind specifically to the target DNA or RNA and capture it.

3. Apply a magnetic field to the tube to separate the beads and bound target DNA or RNA from the unbound material. Remove the unbound material.

4. Wash the beads several times with a wash buffer to remove any residual contaminants.

5. Add the PCR reaction mix to the beads containing the captured target DNA or RNA. The reaction mix should contain Taq polymerase enzyme, dNTPs, buffer solution, and the PCR primer sets specific for the target sequence.

6. Run the PCR according to the specific parameters for your PCR machine.

7. Analyze the PCR products by running them on an agarose gel. The size of the products should match the expected product size for the target sequence.

It is important to optimize the reaction conditions for each primer set and PCR cycle to ensure successful amplification of the target sequence. Additionally, it is important to use appropriate controls to confirm the specificity of the PCR products, such as a negative control without DNA/RNA template and positive controls with known target DNA/RNA.