Biology · Introductory biology · Worked example
Find the PCR product from two primers
The top strand of a template reads 5′-CCTAGATCGT ACCATTGGCA TCGACTAGGC TTCACGGGAT-3′, 40 bases shown in blocks of 10. The forward primer is 5′-GATCGTACCA-3′ and the reverse primer is 5′-CGTGAAGCCT-3′. Find the product and its length, and the number of copies 30 ideal cycles make from one template.
Find the forward primer
The forward primer has the same sequence as the top strand, read 5′→3′. GATCGTACCA appears at bases 5–14, so the product starts at base 5.
Reverse-complement the reverse primer
The reverse primer binds the top strand, so look for its reverse complement. Complementing CGTGAAGCCT base by base gives GCACTTCGGA, and reading that backward gives AGGCTTCACG.
Find it on the top strand
AGGCTTCACG appears at bases 27–36, so the product ends at base 36.
Count the product length
The product runs from base 5 through base 36, both primer sites included: GATCGTACCA TTGGCATCGA CTAGGCTTCA CG.
Count the copies
Each ideal cycle doubles the number of copies, so 30 cycles multiply one template by 2³⁰, about 1.07 billion. Real reactions fall short of this as they plateau.
Result
The product is the 32 bp stretch from base 5 to base 36; 30 ideal cycles make 2³⁰, about 1.07 billion, copies of it from one template.
Your turn
With the same template and reverse primer, the forward primer is changed to 5′-CCTAGATCGT-3′. How long is the product?
Show the answer and explanation
36 bp, from base 1 to base 36.
CCTAGATCGT matches bases 1–10, and the reverse primer still ends the product at base 36, so the product has 36 − 1 + 1 = 36 bases.
Keep exploring
The PCR tool opens with this template and primer pair, marks both binding sites and shows the 32 bp product. It also warns that these 10-base teaching primers are shorter than laboratory primers, which usually have 18 to 25 bases.
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