POLYMERASE CHAIN REACTION (PCR)
Polymerase Chain Reaction is a technique to make multiple copies of target DNA sequence and make it applicable for the downstream processes.
PCR allows the selected DNA region to be amplified and purified from the remainder genome. The key ingredients of PCR are Taq Polymerase, primers, template DNA (target DNA), and nucleotides (A, G, T, C). The primers are the oligonucleotides that are designed so that they can attach to the desired section of the genome and the polymerase can start adding the nucleotides. However, the process is controlled by different temperatures which are divided into the following 3 steps:
- Denaturation (96°C): At this high temperature, the double-stranded helix denatures to form a single-stranded template.
- Annealing (55- 65°C): In this step, the lowering of the temperature allows the primer to anneal to the template DNA.
- The extension (72°): Again, raising the temperature, allows the Taq polymerase to add the nucleotides and in turn extend the primers, synthesizing a new strand.
This cycle is continued multiple times to create multiple purified copies of the target DNA. After PCR, the strands can be studied by separating using Gel Electrophoresis, DNA sequencing, etc.
- DNA Fingerprinting
- Mutation screening
- Drug discovery
- Pre-natal diagnosis
REAL-TIME POLYMERASE CHAIN REACTION (qPCR)
Real-time PCR has proven to become a very important tool for measuring gene expression. This technique simply means collecting the data throughout the PCR process as it happens, in real-time. Thus, this technique helps to combine amplification and detection at the same time. In order to achieve the detection, the PCR product is needed to be tagged using fluorescent dyes. Comparing the product concentration to the florescent intensities gives us the final result.
Real-time PCR uses RNA as the starting material. The RNA is converted to complementary DNA and this process is called reverse transcriptase. The next is using florescent receptors which are of two types: SYBR green and Taqman probes. The remaining process remains similar to PCR. For both the probes, the amount of fluorescence in a sample is detected and plotted against the cycle number. This means ideally the PCR product would be proportional to the amount of fluorescence. The time point at which the fluorescence reaches a defined threshold becomes relative to the gene expression.
- Gene expression
- Examination of transcript variation
- cDNA templates for cloning and sequencing
Molecular Biology is the study of structures, composition, and interactions of nucleic acids and proteins that carry out essential biological processes for the maintenance and functioning of the cell.
The key members of this study are DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins involved in biosynthesis. Out of the 1000 million species living on Earth, each one is different, yet each reproduces itself faithfully. The parent organism in each species transfers extraordinarily detailed information about its composition to the child organism. This phenomenon of HEREDITARY is the central definition of life. After years of evolution, it was discovered that the nucleic composition meaning RNA, DNA is responsible for this phenomenon.
Every cell in the human body has the same DNA. It is the genetic material that makes every person unique. DNA is a double helix structure formed by four chemical bases called nucleotides: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T) which are attached to a sugar-phosphate backbone. An important property of DNA is its ability to replicate itself. This is critical during cell division so that the old copy can create new copies and pass the information forward.
|DNA/RNA Extraction||Treated centrifuge tube,1.5 ml sterile microcentrifuge tube, Deep well microplate (96/384 well, 1 / 2 mL), Centrifuge, water bath|
|DNA Electrophoresis||Gel tank (horizontal/vertical), Agarose gel, DNA Ladders, DNA stain, Transilluminator (for imaging)|
|PCR||PCR plate/tube/strip, Thermocyclers, Taq polymerase with buffer, Sealing films, mats, or caps|
|qPCR||qPCR Plate, thermocyclers, qPCR Mastermix, sealing films|