TP21 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Automatic assembly of PCR products with Web PCR

Assembly of PCR products from the sequences of primers and template sequence is possible using a sequence editor, but it is tedious and error prone. The pydnaweb web pcr service can do the simulation automatically:

This service can simulate the outcome of PCR given the sequence of the primers and of the template. Web pcr accepts pasted sequences in FASTA or Genbank formats.

Important

The last sequences is assumed to be the template, all other sequences are assumed to be primers.

This means that you can add more than two primers if you like.

Copy and paste the three sequences below , paste into the web pcr window and click "submit".


>2_3CYC1clon
CGATGTCGACTTAGATCTCACAGGCTTTTTTCAAG

>1_5CYC1clone
GATCGGCCGGATCCAAATGACTGAATTCAAGGCCG

LOCUS       YJR048W__Chr_10_        2330 bp ds-DNA     linear       05-JUN-2012
DEFINITION  .
ACCESSION   
VERSION     
SOURCE      .
  ORGANISM  .
COMMENT     >YJR048W  Chr 10   from 525335 to 527664  
COMMENT     ApEinfo:methylated:1
FEATURES             Location/Qualifiers
     misc_feature    959..1003
                     /label=New Feature
     misc_feature    1327..1372
                     /label=New Feature(1)
ORIGIN
        1 GAGGCACCAG CGTCAGCATT TTCAAAGGTG TGTTCTTCGT CAGACATGTT TTAGTGTGTG
       61 AATGAAATAG GTGTATGTTT TCTTTTTGCT AGACAATAAT TAGGAACAAG GTAAGGGAAC
      121 TAAAGTGTAG AATAAGATTA AAAAAGAAGA ACAAGTTGAA AAGGCAAGTT GAAATTTCAA
      181 GAAAAAAGTC AATTGAAGTA CAGTAAATTG ACCTGAATAT ATCTGAGTTC CGACAACAAT
      241 GAGTTTACCA AAGAGAACAA TGGAATAGGA AACTTTGAAC GAAGAAAGGA AAGCAGGAAA
      301 GGAAAAAATT TTTAGGCTCG AGAACAATAG GGCGAAAAAA CAGGCAACGA ACGAACAATG
      361 GAAAAACGAA AAAAAAAAAA AAAAACACAG AAAAGAATGC AGAAAGATGT CAACTGAAAA
      421 AAAAAAAGGT GAACACAGGA AAAAAAATAA AAAAAAAAAA AAAAAAAGGA GGACGAAACA
      481 AAAAAGTGAA AAAAAATGAA AATTTTTTTG GAAAACCAAG AAATGAATTA TATTTCCGTG
      541 TGAGACGACA TCGTCGAATA TGATTCAGGG TAACAGTATT GATGTAATCA ATTTCCTACC
      601 TGAATCTAAA ATTCCCGGGA GCAAGATCAA GATGTTTTCA CCGATCTTTC CGGTCTCTTT
      661 GGCCGGGGTT TACGGACGAT GGCAGAAGAC CAAAGCGCCA GTTCATTTGG CGAGCGTTGG
      721 TTGGTGGATC AAGCCCACGC GTAGGCAATC CTCGAGCAGA TCCGCCAGGC GTGTATATAT
      781 AGCGTGGATG GCCAGGCAAC TTTAGTGCTG ACACATACAG GCATATATAT ATGTGTGCGA
      841 CGACACATGA TCATATGGCA TGCATGTGCT CTGTATGTAT ATAAAACTCT TGTTTTCTTC
      901 TTTTCTCTAA ATATTCTTTC CTTATACATT AGGACCTTTG CAGCATAAAT TACTATACTT
      961 CTATAGACAC ACAAACACAA ATACACACAC TAAATTAATA ATGACTGAAT TCAAGGCCGG
     1021 TTCTGCTAAG AAAGGTGCTA CACTTTTCAA GACTAGATGT CTACAATGCC ACACCGTGGA
     1081 AAAGGGTGGC CCACATAAGG TTGGTCCAAA CTTGCATGGT ATCTTTGGCA GACACTCTGG
     1141 TCAAGCTGAA GGGTATTCGT ACACAGATGC CAATATCAAG AAAAACGTGT TGTGGGACGA
     1201 AAATAACATG TCAGAGTACT TGACTAACCC AAAGAAATAT ATTCCTGGTA CCAAGATGGC
     1261 CTTTGGTGGG TTGAAGAAGG AAAAAGACAG AAACGACTTA ATTACCTACT TGAAAAAAGC
     1321 CTGTGAGTAA ACAGGCCCCT TTTCCTTTGT CGATATCATG TAATTAGTTA TGTCACGCTT
     1381 ACATTCACGC CCTCCTCCCA CATCCGCTCT AACCGAAAAG GAAGGAGTTA GACAACCTGA
     1441 AGTCTAGGTC CCTATTTATT TTTTTTAATA GTTATGTTAG TATTAAGAAC GTTATTTATA
     1501 TTTCAAATTT TTCTTTTTTT TCTGTACAAA CGCGTGTACG CATGTAACAT TATACTGAAA
     1561 ACCTTGCTTG AGAAGGTTTT GGGACGCTCG AAGGCTTTAA TTTGCAAGCT TCGCAGTTTA
     1621 CACTCTCATC GTCGCTCTCA TCATCGCTTC CGTTGTTGTT TTCCTTAGTA GCGTCTGCTT
     1681 CCAGAGAGTA TTTATCTCTT ATTACCTCTA AAGGTTCTGC TTGATTTCTG ACTTTGTTCG
     1741 CCTCATGTGC ATATTTTTCT TGGTTCTTTT GGGACAAAAT ATGCGTAAAG GACTTTTGTT
     1801 GTTCCCTCAC ATTCCAGTTT AGTTGTCGAC TGATACTGTT AATAAACTCA TCGGGCGAGG
     1861 CTTCCACGGT TGGAAAAGCA TATGGGCTGG CGCATATGGT TATAAAATCA CCTTTTTGCA
     1921 ATTCAATTCT ATCTTTCCCA TCAAAAGCCG CCCATGCTGG AGCCCTTGAC TTCATCGAGA
     1981 CTTTCACTTT TAAATTTATA CTTTCTGGTA AGATGATGGG TCTGAAACTC AATGCATGTG
     2041 GACAAATGGG TGTTAAAGCG ATTGCATTGA CGGTTGGGCA TACCAATGAC CCACCTGCAC
     2101 TCAAAGAATA GGCCGTGGAC CCAGTCGGAG TAGCAGCAAT CAGTCCGTCC GCCTGCGCAA
     2161 CGGTCATTAA TGAGCCGTCA CCATACAATT CTAACATGGA TAGAAAAGGA CTTGGACCAC
     2221 GATCGATGGT CACTTCGTTC AAAATGTGGT GTGTGCTTAG TTTTTCCACC ACACATATTT
     2281 TCTTCCCCGT GTTTGGGTCT ACTTCAGGGC GGTGTCTACG ATAAATTGTG 
//

You should now get the following output below:

The simulation found one potential PCR product of 359 bp.

The report contain a figure showing how the two primers bind and two suggested PCR programs, one for Taq polymerase and one for a certain class of proofreading polymerases.

Question 1:

Simulate PCR using the three sequences below:


>fp
gctactacacacgtactgactg

>rp
tgtggttactgactctatcttg

>template
atcgtatcgctactacacacgtactgactgcagcatctgacgtaattctactagctgatctccaagatagagtcagtaaccacatgatgcatc

The partial seguid of the PCR product is ldseguid=CM6_tO, what is the complete checksum?

Question 2:

This is an individual question for each student. Go to the Google spreadsheet for this exercise. You should find your name in the leftmost column. Three columns called primer1, 2 and template contains DNA sequences representing primers and a template.

Your task is to use the WebPCR service to simulate the PCR. Put your result in the indicated cell for forward primer (product). Please answer with a raw DNA sequence as indicated for the first example student "Max Maximus".

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