Designing PCR Primers for CFTR Gene Amplification

What is the importance of proper primer design in PCR amplification?

1) Ensuring specificity of the amplified DNA sequence
2) Facilitating successful amplification and detection of sequence variations
3) Both 1 and 2
4) None of the above

Final Answer: The question is about designing PCR primers for amplifying a specific DNA sequence within a gene. Primers must be complementary to the target region and are assessed for specificity using tools like Primer-BLAST. Proper design is crucial for successful amplification and detection of sequence variations.

Explanation:

The subject of the question is about primer design for Polymerase Chain Reaction (PCR) in the context of genetic research, particularly involving the CFTR gene. Primers are short DNA fragments that are complementary to the 3'-ends of the strands of the target DNA which you want to amplify. In designing primers for PCR, it is imperative that the primers are specific to the sequence of interest to ensure that only the desired DNA fragment is amplified. This is achieved by using tools like Primer-BLAST, which combines primer design software with a BLAST search to confirm primer specificity. For the amplification of a target DNA sequence that is part of a protein-coding exon of the CFTR gene, the primers chosen must bracket this specific exon sequence.

For detecting length polymorphisms in simple sequence repeat (SSR) regions, primers must be designed to flank the repetitive sequences, enabling PCR to amplify the region and allowing subsequent analysis to determine the length variations among individuals.

In the context of a mutation, both primers must align with the desired change if creating a mutated sequence is the goal. When considering exon-exon junctions for RT-PCR, primers are ideally positioned in different exons to differentiate cDNA amplification from any potential genomic DNA contamination.