Exploring PCR Amplification and Gel Electrophoresis: A Comprehensive Laboratory Study

1. Objectives

Polymerase Chain Reaction (PCR) Amplification

• Gain proficiency in performing PCR to amplify a specific DNA sequence.
• Acquire knowledge about extracting DNA from hair follicles.
• Understand the functionality of a thermal cycler.
• Comprehend the principles of PCR and its similarity to DNA replication processes.

Gel Electrophoresis of Amplified PCR Samples

• Obtain practical experience in setting up and conducting gel electrophoresis to separate DNA fragments.
• Learn proper techniques for handling agarose gel.
• Visualize DNA bands in the gel through staining techniques.

2. Abstract

This experiment focuses on utilizing polymerase chain reaction (PCR) to amplify a specific DNA chain, specifically the PV92 region of chromosome 16, aiming to determine the presence of the Alu sequence within that region.

PCR will be employed to amplify the target DNA sequence extracted from hair follicles.

The experiment involves three main steps of PCR:

1. Denaturation Step
2. Annealing Step
3. Extension Step

Results will be analyzed by performing gel electrophoresis on the amplified DNA sequence. The outcomes will fall into three categories based on the DNA size of the PCR product: Genotype | DNA Size of PCR Product (bp) Homozygous (+/+) | 941 Homozygous (-/-) | 641 Heterozygous (+/-) | 941-641 Table 2.

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1 - Classification of Genotype versus the DNA size of PCR product

Polymerase Chain Reaction (PCR) is a method employed to amplify specific segments along a DNA strand, with the capability to amplify up to 10 kilo base pairs. This process involves thermo cycling, comprising three essential steps: denaturation, primer annealing, and extension of new DNA strands.

During denaturation, the mixture is subjected to high temperatures, leading to the separation of the parent strand DNA.

Subsequently, in the annealing process, the mixture is cooled, and primers actively seek complementary sequences in the DNA, forming hydrogen bonds. The extension process follows, involving a reheating to 72°C, the optimal temperature for Taq polymerase to synthesize new complementary DNA strands, initiating from the 3’-OH ends of the primers. These three steps are iterated through 40 cycles, as detailed in Section 7, Question 4.

3.2 Gel Electrophoresis of Amplified PCR Samples

Figure 3.1 – Gel Electrophoresis

In this experiment, Gel Electrophoresis was employed to identify target sequences. Gel Electrophoresis operates based on the principles of size and charge, facilitating the separation of fragments. Upon loading samples into the gel wells, an electric current is applied through electrodes at both ends. As DNA fragments carry a negative charge, they migrate across the gel, being attracted to the positive end.

The rate of movement is determined by fragment size, with larger fragments encountering more resistance and moving more slowly through the gel matrix. This allows us to discern the size of DNA fragments and identify the presence of the Alu insertion.

PCR Amplification:

1. 200 μL InstaGeneTM matrix plus protease
2. 1 x tube of complete master mix (with primers) on ice
3. 2 x strands of hair from the same person
4. Foam micro test tube holder
5. Vortex mixer
6. Stopwatch timer
7. PCR tube
8. Capless micro test tube
9. P-20 Micropipet
10. Pipet Tips
11. Screwcap tube
12. Water bath at 56 °C
13. Water bath at 100°C
14. Centrifuge
15. Mycycler Thermal Cycler

Gel Electrophoresis:

1. Student's PCR sample
2. 10 μL PV92 XC DNA loading dye
3. 275 mL of TAE electrophoresis buffer
4. 10 μL MMR (DNA standard)
5. 10 μL PV92 homozygous (+/+) control sample
6. 10 μL PV92 homozygous (−/−) control sample
7. 10 μL PV92 heterozygous (+/−) control sample
8. 120 mL Fast BlastTM DNA stain (100x)
9. 1.5 - 2 L warm tap water

Procedures:

For this part of the experiment, collect 2 strands of hair for each group to prepare the DNA template for the subsequent lab session. Utilize the hair bulb and InstaGene matrix plus protease for DNA template extraction from the hair follicle.

Step 2a: Obtain a PCR tube containing 10 μL of yellow PCR master mix. Step 2b: Use a micropipette to transfer 10 μL of the DNA template from the supernatant in the screwcap tube into the PCR tube containing the master mix. Step 2c: Ensure thorough mixing by pipetting up and down 2 to 3 times, avoiding the formation of air bubbles, especially at the bottom of the PCR tube. Step 2d: Place the PCR tube in the MyCycler thermal cycler. Step 2e: Allow 40 cycles of amplification to take place within the 3-hour timeframe.

Laboratory Calculations and Formulas:

No specific calculations or formulas are explicitly mentioned in the provided information. However, it's essential to be mindful of the volumes being used in pipetting, especially when transferring the DNA template into the PCR tube. Precise and accurate pipetting is crucial for obtaining reliable experimental results.

Table 5.1 - Materials for PCR Amplification

Table 5.2 - Materials for Gel Electrophoresis

The provided laboratory setup and procedures involve essential steps for PCR amplification and gel electrophoresis, utilizing specified materials and equipment. Precise pipetting and adherence to the outlined procedures are crucial for successful experimentation.

1. Necessity of Primers in PCR:
   • To initiate DNA replication, primers are crucial on both sides of the DNA segment to be amplified. DNA polymerase requires a primer with a 3' hydroxyl terminus to start synthesizing a new DNA strand. A single-stranded DNA circle cannot serve as a template for DNA polymerase because the enzyme cannot initiate DNA strand formation. Primers, complementary to the DNA template, define the starting point for replication, attaching to the template before the polymerase can bind to the free 3' end of the primer.
2. Origin of Taq DNA Polymerase Name:
   • Taq DNA polymerase derives its name from Thermus aquaticus, a thermophilic bacterium. Isolated from this bacterium, Taq DNA polymerase is known for its heat resistance, thriving in high-temperature environments like hot springs. Its heat resistance makes it suitable for the denaturation step in PCR.
3. Composition and Functions of Master Mix Components:
   • Nucleotides (A, T, G, C): Essential building blocks of DNA, necessary for DNA polymerases to synthesize new complementary strands.
   • Deoxnucleoside Triphosphates (dNTPs): Adenine, Guanine, Thymine, and Cytosine, raw materials for DNA polymerases.
   • DNA Polymerase: Enzyme reading the DNA template and assembling nucleotides into a new DNA chain.
   • Magnesium Ions: Cofactors required by DNA polymerase for its function in creating the DNA chain.
   • Oligonucleotide Primers: Fragments of DNA complementary to the 3' ends of the DNA template, serving as the starting point for replication.
   • Salt Buffer: Provides an ionic environment and pH to optimize the reaction and maintain DNA polymerase stability.
4. PCR Amplification Cycle Steps and Reactions:

1. • A complete PCR involves 40 cycles (Table 7.1).
2. Precise Length Target DNA Sequence Amplification Explanation:
   • The precise length target DNA sequence starts amplifying from the third cycle onwards. In the initial cycles, heat denaturation and primer binding occur, but the synthesized strand still contains regions of non-interest. From Cycle 3, the target DNA sequence amplifies, ensuring that only the desired segment is replicated in subsequent cycles. Amplification starts only when the newly synthesized strand contains the target sequence (Figure 7.1).