Q1) Explain the use of the BLAST tool
Q2) What does BLAST do?
Q3) The sequence shown below was isolated from the causative agent of a respiratory infection.:
MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLC
DLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYE
ELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNNAY
PTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIAT
RSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGN
CNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQIETRGLFGAIA
GFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQF
EAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKV
RLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESI
GTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI
Use the BLAST search tool to find out what the infectious agent was?
Q3) The sequence for the Beta Actin gene (ACTB) is given below. Which pair of primers (designed to amplify the entire sequence below) could be used to successfully produce the beta actin PCR product. Circle your answer (note all sequences are written 5’ to 3’).
a) Forward = ggctcacagcgcgcccggctat
Reverse = taaaagtgcacaccttaaaaatga
b) Forward = tatcggcccgcgcgacactcgg
Reverse = tcatttttaaggtgtgcactttta
c) Forward = ggctcacagcgcgcccggctat
Reverse = tcatttttaaggtgtgcactttta
d) Forward = atagccgggcgcgctgtagcc
Reverse = taaaagtgcacaccttaaaaatga
Explain your reasoning
Q4) A pandemic is underway that has infected millions of people worldwide. It is caused by a strain of influenza that has crossed with an avian virus. You must design PCR primers so that reverse transcriptase PCR can be performed to diagnostically test the presence of the virus in patients.
You obtain the following sequence for a gene in the virus. Note that the sequence given below is the same as the mRNA (except that the uridines are shown as thymidines). See the notes below about the ‘polarity’ of RNA viruses.
1 acaaaaacat aatggattcc aacactgtgt caagctttca ggtagactgc tttctttggc
61 atgtccgcaa acgatttgca gaccaagaac tgggtgatgc cccattcctt gaccggcttc
121 gccgagatca gaagtccctg agaggaagag gcaacactct tggtctggac atcgaaacag
181 ctactcgtgc ggggaaacag atagtggagc ggattctgga tgaggaatct gatgaggcgc
241 ttaaaatgcc gacttcacgc tacctaactg acatgactct cgaagaaatg tcaagggact
301 ggttcatgct catgcccaag cagaaagtgg tgggttccct ttgcatcaaa atggaccagg
361 caatgatgga taaaaccgtc atattgaaag caaacttcag tgtgattttt gaccgattag
421 aaaccctaat actgcttaga gctttcacag aagaaggagc aatcgtggga gaaatctcac
481 cattaccttc tcttccagga catactagtg aggatgtcaa aaatgcaatt ggcgtcctca
541 tcggaggact tgaatggaat gataacacag ttagggtctc tgaaactata cagagattcg
601 cttggagaag cagtgatgag ggtgggagac ttccactccc tccaaatcag aaacggaaaa
661 tggcgagaac aattgagtca gaagtttgaa gaaataaggt ggctgattga agaagtacga
721 catagattga aaattacaga aaacagcttc gaacagataa cgtttatgca agccttacaa
781 ctactgcttg aagtggagca agaga
You will use ‘Primer 3’, an online primer design program, to design the primers for your PCR reaction.
Go to the web address https://frodo.wi.mit.edu/primer3/. Copy the sequence above into the first text box. Click on the button which says ‘Pick Primers’ (NB all settings will be left at default).
a) Copy the sequences of the first pair of primers suggested by primer 3 and paste them below (note that the primer 3 output will give you the FIRST choice above the sequence, and then a series of alternative choices listed as 1-4 below the sequence (you should select the first choice that appears above the sequence).
b) What are the characteristics of the primers in terms of length, GC content, Tm (NB – you can use the Tm calculated by primer 3
c) What is the size of the PCR product you would expect?
d) What is ‘primer dimer’?
e) PCR efficiency can be compromised if the primers form internal secondary structure or if the pair of primers form ‘primer dimers’. Put the sequences into the following ‘oligo calculator’ (https://www.sigma-genosys.com/calc/DNACalc.asp ) and press ‘Calculate’. Do the primers produce secondary structure (Sec. Str.) or primer dimer?
f) Would you accept this primer pair as suitable for amplifying the influenza strain? Explain your reasoning.
g) Design and describe a PCR-based experiment to specifically detect the presence of the flu virus genome in mammalian cells using your primers designed in part a). Remember that the viral genome is based on RNA (see attached sheet on the influenza genome). Include the list of controls you would include to ensure that your results are meaningful. NB When thinking about which primer to use in the ‘RT’ step you should consider whether the forward or reverse primer will anneal to the viral genome and which will anneal to the mRNA copy produced in cells.
h) How could you modify the experiment to distinguish between the viral genomic RNA and the copy RNA that is produced following infection of a cell (NB – the sequence given above is the cRNA sequence, i.e. the copy RNA that can be directly translated into protein)?