Final answer:
The key difference between BLASTX and BLASTP lies in the query sequences and databases they match, with BLASTX translating nucleotide queries into protein sequences while BLASTP matches protein queries directly. Additionally, megablast is designed for more similar sequences and uses a larger word size than blastn, potentially explaining fewer, more specific matches. Moreover, an optimal primer is crucial for targeted amplification and is designed considering several parameters for effectiveness.
Step-by-step explanation:
The major difference between BLASTX and BLASTP lies in their respective uses. BLASTX is an algorithm that translates a nucleotide query sequence into all possible protein sequences (six reading frames) and compares them to the protein sequence database. In contrast, BLASTP simply compares an amino acid query sequence against a protein sequence database. Therefore, the differences in the results between these two methods are partly due to the nature of the query sequences and the databases they are compared with.
Concerning the difference between megablast and blastn, they are both nucleotide-to-nucleotide comparison tools, but megablast is optimized for highly similar sequences and uses a larger word size compared to blastn. A difference in word size could explain the variation in the number of matches; a larger word size in megablast could lead to fewer, but more specific, matches compared to the more sensitive blastn that uses a smaller word size and can find more matches, including less similar ones.
The E-value is a critical parameter that helps differentiate between meaningful and random sequence alignments, with values closer to zero indicating more significant alignments. Additionally, gap penalties are used during the alignment process to ensure the highest scoring alignment is identified, and BLAST searches can be performed against specific databases such as the Protein Data Bank (PDB) or the human genome.
When designing primers, sequence choices and optimization are critical. An optimal primer will have properties like the correct melting temperature, lack of secondary structures, and specificity for the target sequence intended for PCR amplification or sequencing.