Question

Completing the table below PCR and gel electrophoresis Key scientific principles which form the basis of the technique: PCR is a cyclic reaction that replicates specific DNA fragments in vitro using the principle of double-stranded DNA replication. A large number of copies of a specific DNA region can be amplified rapidly by thermal cycling. DNA is visualised using gel electrophoresis, fragments are pulled through a gel matrix by an electric current and separated according to sizes. DNA ladder is included to determine the size of the fragments in PCR sample. Necessary controls, standards, and processing. Describe and state the purpose of these. 1. Controls: 2. Standards: DNA Ladder 3. Data processing (e.g. cleaning/trimming, normalisation, etc): Interpretation: List each possible outcome for this technique, and describe how each of these outcomes should be interpreted with reference to controls and/or standards A B C … Follow-up analysis technique: For each of the possible outcomes described in the interpretation section, explain which follow up analyses are needed (where applicable). Use the letter (A, B, C) assigned in the “Interpretation” section to refer to each of these. Limitations:

Answer 1

The principle of the PCR is based on the temperature variations of heating and cooling- thermocycling reaction divided into three steps:

Denaturation- The dsDNA becomes single-stranded at a higher temperature during denaturation. Here hydrogen bonds between two DNA strands break.

Annealing- in The primer binds or anneals to its exact complementary sequence on a DNA during the annealing step. The primer provides a site for the initiation of synthesis.

Extension- Taq DNA polymerase uses the 3’ end of the primer and starts DNA synthesis by adding nucleotides to the growing DNA strand.

All three steps are repeated for 25 to 40 cycles and in each cycle the DNA becomes double.

PCR reagents:

1.Template DNA: The template must be DNA only. Plasmid DNA, bacterial DNA, cDNA, or gDNA can be utilized as a template. The template DNA should be a highly purified DNA that has a purity of around ~1.80 and a quantity of up to 200ng. The DNA works as a substrate for an enzyme when it denatured.

2.PCR Primer: RNA primer governs the replication reaction, normally, however, DNA primers are utilized during PCR instead of RNA primers.

3.dNTPs: Deoxynucleotide triphosphates are artificially synthesized nucleotides which bind to the growing DNA strand. With the help of the Taq DNA polymerase, the dATP, dGTP, dCTP, and dTTP binds at its complementary nucleotides on the growing DNA strand.

4.Taq DNA polymerase: The PCR technique is entirely based on the activity of Taq DNA polymerase. The Taq DNA polymerase settles at the ssDNA- primer junction and utilizes it as a substrate for the catalytic reaction. In the final step of extension, using the substrate it starts dNTP insertion.

DNA Ladder: The DNA ladder is a standard-sized fragment of DNA used to determine the molecular weight of the PCR amplicons. The commercially available DNA ladders come under 50bp, 100bp, 1000bp, and 3000bp form.

After PCR run It will be analyzed using DNA Agarose gel electrophoresis.

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In-gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule.

he DNA molecules separated on the basis of their charge and size.

As the concentration of agarose increases, the size of pore will decreases and vice verse. Therefore, the larger molecules cannot run faster in comparison with the smaller DNA fragments.

Once the electrical current passes through the gel, the DNA starts moving towards the opposite electrode.

A tagged fluorescent dye such as EtBr used to visualize DNA bands. Another dye named DNA gel loading dye monitors the migration of DNA by running ahead of it.

Furthermore, a known molecular weight DNA marker is used to calculate the exact size of DNA.

Once the DNA reached a sufficient distance, the process is stopped to protect the DNA running out of the gel.

Principle of agarose gel electrophoresis

The negatively charged DNA molecules migrate towards the positive charge under the influence of constant current, thus the separation depends on the mass and charge of DNA. The DNA molecules are forced to move through the agarose gel pores. The rate of the migration depends on,

• The strength of the field
• The hydrophobicity of the DNA
• The ionic strength of the buffer
• The size and shape of the DNA
• The temperature of the buffer

Three of the important factors that affect DNA migration rate are,

• Agarose concentration
• Conformation of DNA
• Size of DNA

Component of agarose gel electrophoresis

• Agarose
• Agarose gel electrophoresis equipment
• Agarose gel electrophoresis buffer
• DNA Gel loading dye
• EtBr
• Powerpack

Results interpretation:

A band is a compilation of a DNA fragment of a similar type. The Know molecular marker, known as the DNA ladder is used to determine the fragment size.