Given the following DNA sequence and PCR primers, underline and label the anneal

Question

Given the following DNA sequence and PCR primers, underline and label the annealing locations for the two primers and draw a box around the target sequence

Please underline and label the annealing location for the two primers and draw a box around the target sequence. Please explain how this is done

5’-AGTCGCTATTCGGATATCGCGCTAGTTCGATAGAGCACGAGCTCG-3’

3’-TCAGCGATAAGCCTATAGCGCGATCAAGCTATCTCGTGCTCGAGC-5’

Primer1: GTCGCTATTCG

Primer2: GAGCTCGTGCT

If you can answer these other questions, it will be appreciated but really need to understand the first question. Thank you

If you are using a DNA sequence from a related species to design primers for your species of interest, why is it better to design your primers to complement an exon?

Why is it better to use a 25-base primer than a 10-base primer? Why do you have to have a lower annealing temperature for a 10-base primer?

Explanation / Answer

1). A primer is a DNA strand which is complimentary to a particular portion of the DNA of interest. Since adenine pairs only with thymine and guanine with cytosine, the annealing location can be found by finding the complimentory sites.

Primer 1 GTCGCTATTCG complimentary bases CAGCGATAAGC

Primer 2 GAGCTCGTGCT Complimentary bases CTCGAGCACGA

Hence the annealing locations are

5’-AGTCGCTATTCGGATATCGCGCTAGTTCGATAGAGCACGAGCTCG-3'

Primer 2 GAGCTCGTGCT

3’-TCAGCGATAAGCCTATAGCGCGATCAAGCTATCTCGTGCTCGAGC-5’

GTCGCTATTCG Primer 1

While determining the complimentary strands it has to be taken into account that primers also have 5' to 3' direction and binds to a strand in opposite direction ie. 3' to 5' or vice versa

2). Eventhough related species have similarity in genetic material they are never the same. One is not an exact copy of another. Exons are those parts of DNA which codes for proteins and is the expressed genetic material. A mutation in exon changes genetic characteristics of an individual. Hence it's better to design exons to prevent accidents because even in related species exons may differ.

3) (A) Usually in PCR technology 18 to 25 base primers are preferred because they are small enough for easy annealing and is large enough for ensuring stability. A 10 base primer is not specific enough that is it may pair with a wrong portion of DNA.

(B) Annealing temperature increases when the number of base pairs increase. Hence a 10 base primer has a low annaeling temperature.

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