Sometimes it is helpful to obtain sequence data from more than one gene when ass

Question

Sometimes it is helpful to obtain sequence data from more than one gene when assigning a species identity to a sample. The following is sequence from the Amyrel gene of an unknown insect.

        1 aattccctgg aatagcaatg aaattattgc caagcctact cgcggtagcc ttggcctttg

       61 gcttggtgtc tgcgcagttc gacacccatc agtgggcagg tcgaagcgga atcgtgcacc

      121 tgttcgagtg gagatggaac gacattgcgg atgaatgcga gcggtttttg gccccgagag

      181 gctatgccgg tgtccaggtt tcgccaccaa cggagaacgt aatcatcgct ggacgacctt

      241 ggtgggagcg ttaccagcca gcttcctatc atctgaacac ccgctccggt actgaagccg

      301 aattcgcaag catggtacgg cgctgtaatg acgttggagt gcgaatctac gtggacatcg

      361 ttatcaacca tatggccgcc gtttccggac aaggtactgg aggatcgact gttaatggat

      421 tgaacttccc tgcagtcccc tacggaccga acgacttcaa tcctccttgt gaaatctacg

      481 actacaacaa ccgctatcag gtgcgtaact gctggctggt cggccttcca gatttggccc

      541 taggaaacga atggcctcgt tggcgagtca tcgatttgat gaacaagtgt atcaactatg

      601 gcgttgccgg attccgtgtt gatgctgtaa agcacatgtg gcctgcagat ttggaattca

      661 tctacggcaa cctggttact ctgaacaccg accatggatt cccggcggga gcgagggctt

      721 tccttacgca ggaggtcatt gatttgggca acgaagcgat ctccagcacg gaatacactc

      781 acctgggaac ggttaccgag ttcaaacatt ctgccgaaat cggacgagta ttctacggac

      841 gcgatagact agcccacctg tcgaactggg gcgaaggatg gggattcctt ccgtcgcact

      901 tggcactggt gttcgtcgac aatcacgata accaacgagg tcacggagct ggaggcgaca

If we wanted to PCR and sequence part of this gene we would first need to design forward and reverse primers to amplify a stretch of sequence by PCR.

a)      Give the sequence (written out from 5' to 3') of a 22bp forward primer and a 22bp reverse primer that could be used to amplify ~300bp of this sequence.

b)      If we used the primers from (a) exactly how many base pairs would the PCR product be?

c)       When you conduct the PCR and run the reactions on a gel you are pleased to see a bright band of the expected size on your gel. However, you also see two faint larger bands. What is a possible explanation for why these bands are being produced by the PCR?

d)      Using BLAST to help you, what species is this unknown insect most likely to be?

Explanation / Answer

A) Several set of primers can be designed to amplify a 300bp sequence from this gene. I have provided with 1 set here.

5'-tc gacacccatc agtgggcagg-3'

5'-cg ttatcaacca tatggccgcc-3'

while designing a primer, several things should be kept in mind. The primer should be designed to be as unique as possible. That is to say that the oligonucleotide sequence should not be complementary to any other region in any other gene present in the organism.

To assure this, often the primer sequence, as well as genome sequence of the organism, are aligned using sequence alignment tools of NCBI blast.

Secondly, the primer length should be optimum. It shouldn't be too short such that there is nonspecific binding nor too long such that the melting temperature and the annealing time is prolonged. Moreover, the GC content of the primer should be between 40-60%.

The primer characteristics can be calculated by pasting the sequence in several online tools such as oligoCalc. Melting temperature of both forward and reverse primer should be similar and must not have a difference of more than 5 degrees.

The forward primer is chosen directly from the sequence (as it is provided in the 5' to 3' direction) whereas, the reverse primer is chosen from the sequence and its reverse complement is used. This can be written manually [ write the complement sequence and then reverse it] or an online tool called Emboss : RevSeq, can be used for this.

B) 302 bp

C) the faint larger sized bands are due to non-specific amplification of the template. The designed primers can somehow have a stretch of the complementary region within the rest of the gene. This might result in binding of primer at various region during the gene and amplification initiation.

D) open NCBI blast nucleotide tool. paste the sequence in the window in appropriate format (Fasta format involves '>' followed by the sequence from the next line) and blast it against all the nucleotide sequence containing database.

The sequence is highly similar to Aedes aegypti amylase gene.

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