Using NCBI Primer BLAST software, design a set (pair) of primers for each of the following genes, using the given parameters. Use the Primer Design tutorials and notes on the Hub for step-by-step guidance. FOLLOW INSTRUCTIONS BELOW CAREFULLY!
(1) SERPINA1 (human, 14q32.13)
o Search parameters:
? PCR product size should be between 200 and 500 bp
? Compare against human genome
? All other parameters stay at default
o Submit the following:
? List the primers sequences for the first (best) primer pair (indicated which is forward and reverse)
? List the Tm for each primer in that set
? Predict the optimum annealing temperature for the primer set
? List the amplicon size
Question 1 of 5
What follows is a fill in the blank question with 2 blanks.
For SERPINA1, the first (best) primer set includes the following sequences:
Forward Primer: 5′ Blank 1. Fill in the blank, read surrounding text. 3′
Reverse Primer: 5′ Blank 2. Fill in the blank, read surrounding text. 3′
• Question 2 of 5
• What follows is a fill in the blank question with 3 blanks.
• For SERPINA1, the melting temperatures (Tm), according to Primer Blast, for each primer are as follows:
• Forward primer: Blank 1. Fill in the blank, read surrounding text. degrees C
• Reverse primer: Blank 2. Fill in the blank, read surrounding text. degrees C
• The optimal PCR annealing temperature for the primer set would be: Blank 3. Fill in the blank, read surrounding text. degrees C
• Question 3 of 5
• What follows is a fill in the blank question with 1 blanks.
• For SERPINA1, the amplicon size of the first primer set is Blank 1. Fill in the blank, read surrounding text. bp.
(2) Transthyretin (human, 18q12.1)
o Search parameters:
? PCR product size should be between 70 and 1000 bp
? Compare against human genome
? Change primer specificity stringency to:
• Primer must have at least 6 total mismatches to unintended targets, including at least 3 mismatches within the 5 bps at the 3’ end
• Ignore targets that have 9 or more mismatches to the primer
o Submit the following:
? List the primer sequences for the first (best) primer pair (indicated which is forward and reverse)
? List the amplicon size of the intended product of the first primer set
? List the amplicon sizeof the 3 most likely unintended (non-specific) targets for this primer set
For transthyretin, the first (best) primer pair has the following sequences:
Forward primer: 5′ Blank 1. Fill in the blank, read surrounding text. 3′
Reverse primer: 5′ Blank 2. Fill in the blank, read surrounding text. 3’
The amplicon size is Blank 3. Fill in the blank, read surrounding text. bp
Question 5 of 5
What follows is a fill in the blank question with 3 blanks.
There were several unintended products for the transthyretin gene primer set.
The amplicon size of the first unintended product, zinc finger protein, was Blank 1. Fill in the blank, read surrounding text. bp.
The amplicon size of the second unintended product, netrin receptor DCC, was Blank 2. Fill in the blank, read surrounding text. bp.
The amplicon size of the third unintended product, integrin alpha-9 precursor, was Blank 3. Fill in the blank, read surrounding text. bp.