Molecular Biology

Procedure of RFLP Technique:

1. DNA Extraction: Extract genomic DNA from the sample.
2. Digestion with Restriction Enzymes: Cut the DNA with specific restriction enzymes that recognize particular sequences, producing fragments of varying lengths.
3. Gel Electrophoresis: Separate these fragments on an agarose gel based on size.
4. Transfer to Membrane: Transfer the separated DNA fragments to a nylon membrane by Southern blotting.
5. Hybridization with Probe: Hybridize the membrane with a labeled DNA probe that binds to a specific DNA sequence.
6. Detection: Visualize the pattern of DNA fragments by autoradiography or fluorescence.

Different individuals have unique patterns of restriction fragments due to variations in their DNA sequences, making RFLP useful for DNA fingerprinting. Use in Forensic Sciences: RFLP is used in forensic investigations to identify individuals based on their unique DNA patterns. It helps in crime scene analysis by matching DNA samples from suspects with evidence, establishing identity or paternity, and solving cases where other evidence is unavailable.

The enzyme used in Sanger’s method of DNA sequencing is DNA polymerase. This enzyme synthesizes a new DNA strand by adding nucleotides complementary to the template strand. It incorporates both normal deoxynucleotides (dNTPs) and chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis, allowing sequence determination based on chain termination.

Using ddNTPs conjugated with fluorescent molecules in automated DNA sequencing offers several advantages:

• Simultaneous detection: Different fluorescent dyes attached to each ddNTP allow all four nucleotides to be identified in a single reaction mixture.
• High throughput and speed: Automated systems detect fluorescence as DNA fragments pass through a laser detector, enabling rapid sequencing.
• Increased accuracy: Fluorescence signals provide clear, precise identification of terminating bases, reducing errors.
• Safety and convenience: Eliminates the need for radioactive labeling and manual gel reading.
• Automated data analysis: Fluorescence data can be processed by software for quick and accurate base calling.

In Sanger’s DNA sequencing method, primers are short single-stranded DNA sequences that provide a starting point for DNA polymerase to begin synthesis of the new DNA strand.

• Primers anneal to the single-stranded DNA template at a specific site, ensuring that DNA synthesis starts at a known location.
• DNA polymerase can only add nucleotides to an existing 3-OH group; primers provide this 3-OH group.
• Without primers, DNA polymerase cannot initiate DNA strand synthesis, so sequencing would not proceed.

Thus, primers are essential for initiating the DNA replication process during sequencing.

Cause: Chronic Myelogenous Leukemia (CML) is caused by a genetic abnormality known as the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11). This translocation creates a fusion gene called BCR-ABL that produces an abnormal tyrosine kinase protein. This protein leads to uncontrolled proliferation of myeloid cells causing leukemia. Technique to know disease status: The status of CML is monitored using the Fluorescence In Situ Hybridization (FISH) technique. FISH detects the presence of the BCR-ABL fusion gene and the Philadelphia chromosome in patient cells, helping in diagnosis and monitoring treatment response.

PCR (Polymerase Chain Reaction) is a technique used to amplify DNA. In one cycle of PCR, the number of DNA molecules doubles because each DNA strand serves as a template for replication. Starting with two DNA molecules, after one cycle, the number doubles to four molecules.

• Option C is correct because 2 molecules become 4 after one PCR cycle.
• Options (A) and (B) represent DNA quantities after multiple PCR cycles.
• Option (D) is incorrect because the number does not stay the same or decrease.

Thus, the correct answer is (C) Four.