Design a forward and reverse primer ATGTCTAAAACTGGAGGCAAGATTAGTTTCTACGAA GACCGAA

Question

Design a forward and reverse primer

ATGTCTAAAACTGGAGGCAAGATTAGTTTCTACGAA

GACCGAAATTTTCAAGGCCGCCGCTATGACTGCGACTGCGACTGCGCAGACTTCCGCTCGTACCTAAGTCGCTGCAACTC

CATTAGAGTAGAGGGAGGCACCTGGGCTGTGTATGAAAGGCCCAACTTCTCCGGGCACGCGTACATCTTACCCCAGGGCG

AGTACCCTGAATACCAGCGTTGGATGGGCCTTAATGACCGCCTGGGCTCTTGCCGAGCTGTTCATCTGTCTAGCGGAGGC

CAGGCTAAGATTCAGGTCTTTGAAAAGGGCGACTTCAACGGTCAGATGTACGAAACCACGGAAGACTGTCCTTCTATCAT

GGAGCAGTTTCACCTGCGAGAGATTCATTCCTGTAAGGTGGTAGAAGGCACCTGGATTTTCTATGAGCTACCCAACTACC

GTGGCAGACAGTATCTTCTGGATAAGAAGGAGTACCGGAAGCCCGTCGATTGGGGTGCGGCATCCCCCGCTATCCAGTCG

TTCCGCCGCATTGTGGAGTGAGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGC

GGCCGCTCGAGCAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAG

CATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATAATTCTTGAAG

ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTT

TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA

CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCACATTTCCGTGTCGCCCTTATTCCCTTTTTG

CGGCATTTGCCTTCCTGTTTTGCTCACCCAGAAACGNTGGNNAAAGTAAAAGATGCTGAAGATCAGTTGGGTGNNCNNNG

Explanation / Answer

A primer is a short piece of DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis. It is required for DNA polymerases to carry out DNA replication. This enzyme can add nucleotides to the 3’ end of the existing strand of DNA.

Things to keep in mind while designing primers:

1. Primer length: Usually it can be between 18-22 nucleotides. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature.

2. Primer melting temperature (Tm): is the temperature at which one half of the DNA duplex will dissociate to become single stranded. Primers with melting temperatures in the range of 52-58o C generally produce the best results.

3. Primer Annealing Temperature: is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high annealing temperature may lead to insufficient primer and template hybridization and too low annealing temperature may lead to non-specific amplification caused by a high number of base pair mismatches.

4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer should be 40-60%.

5. Primer Secondary Structures: Primers should not have any secondary structures such as hairpins, self dimers and cross dimers.

Forward primer:

It can be the first 18-22 nucleotides of the given sequence.

ATGTCTAAAACTGGAGGCAAG

Reverse primer:

It is the reverse complement sequence of the last 18-22 nuclotides

CGGCATTTGCCTTCCTGTTTTGCTCACCCAGAAACGNTGGNNAAAGTAAA

AGATGCTGAAGATCAGTTGGGTGNNCNNNG

TCTACGACTTCTAGTCAACCCACNNGNNNC (Complement)

CNNNGNNCACCCAACTGATCTTCAGCATCT (reverse complement)

Reverse primer:

CNNNGNNCACCCAACTGATC

Since N can be any one of the four bases (A, T, C or G) you may have design some degenerate primers.

There are many sotwares available to design primers for the given sequeneces. one example is Primer3 (the below link)

http://simgene.com/Primer3

Degerate primers:

A degenerate primer contains a mix of bases at one or more sites. You can design degenerate primers in Geneious by using either a sequence containing ambiguous bases or an alignment as the template and checking the Allow degeneracy box. The degeneracy value that you specify is the maximum number of primers that any primer sequence is allowed to represent. For example, a primer which contains the nucleotide character N once (and no other ambiguities) has a degeneracy of 4 because N represents the four bases A,C,G and T.

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