Molecular Biology Review Questions:
The following shows the first 370 bases of the cDNA sequence for the
Drosophila melanogaster amnesiac (amn) gene. Only the coding strand is
shown.
GAAGGGGTGC
AAAACAATAC
AACGGTTACT
AGTTATTTCC
ACCCATTAAT
TACTCCTTTT
GGTGGCTAAG
TTTACAGGAT
TGAAAAGGAT
GGAGAGAGAG
TTGAGAGAAA
TGTGTGTGTG
GCACATCCTA
AATACCAACG
TTAGTGCTCA
CCAATGCAAT
AATAACGGAT
TCTACCCTTA
TATGTACACT
CCAGATAAAA
TAAGAACTAC
AGAGAGGGTT
ATAAGGATAA
CACGTCCTGA
AGTGCGCAAA
GCAGATTTGG
TTAATGTTTT
ACGTTAAGAT
GATTCGGAAA
CTACCGTATA
GGCGAAAATA
CGCGCAGATA
AGATTCAAAA
TAGGTGGAGG
ACAAGCCGAA
CCGCGTGCAC
AGCCATGATT
TCATTCAGGC
TATGTGCTTT
ACTCATCTAC
TCGCATACGA
CGTAATAACA
CCTTTCAGCA
AAAATGTTAG
GGCTGCTCCT
CAAGTCGGCA
GGATATGGGA
ACACACGCCG
GCAAGCTTTT
AGGCAAATTG
AATAGATACG
TAAGAGACGA
AAACGCCTTA
ATAATGAGCC
AGCATAACCA
GCAGTGACAT
TTTCTTTCAG
GGGGAGCAGG
CGGCACTGTT
CGGACTCGGA
TCAAATAACT
CTACTATTTT
ATTTTTATTT
GTTGTATATT
AGAGACGATT
TAGCTTCCTT
CTGTTGCAGA
TGTGAGAGAG
AAGCATGAAA
GCTTCGCAAG
Give the sequence of the 20 base pair forward and reverse primers that could be
used to sequence this entire gene segment.
Forward primers are always the coding sequence: 5’ GAAGGGGTGC GCACATCCTA 3’
Reverse primers are the reverse and compliment of the coding strand so:
5’ CGGCGTGTGT GTGCACGCGG 3’
If you were to use the forward primer as your sequencing primer, how long would
the resulting sequence be (assuming that all bases were successfully sequenced)?
370 bases minus the 20 base primer (the sequence starts with the first base after
the primer) = 350 bases
Give the first 10 bases of sequence that you would expect to get from the reverse
primer.
5’ TCAGGACGTG 3’
What does “N” mean in the results of a sequencing reaction?
The the program is unable to call a base…it could be any of the 4 bases.
Be able to discuss how next generation sequencing differs from Sanger (dideoxy)
sequencing at both a chemical and a data level. How do each of the types of
sequencing that we studied work, how long is each sequence (on average), and how
much does it currently cost to generate sequences in each of these manners?
See the class notes.
What is a contig…and how do the contigs generated by next gen sequencing differ
from those generated by Sanger sequencing?
Contigs are DNA sequences generated by overlapping sequences from independent
sequencing reactions. Next Generation sequencing generates much smaller DNA
sequence reads, so more sequencing reactions are needed to generate a contig than
in Sanger sequencing.