Assignment 3 - March 12, 2018
Verifying the structure of DNA constructs
using restriction digests
This assignment is worth 5% of
the course grade.
Due by 11:59 pm, Wednesday March 21
It is not enough to go through the steps of creating DNA
constructs using standard cloning procedures. Each construct must
be verified to ensure that the resultant DNA is actually what we
intended it to be. While in principle this could be done by DNA
sequencing, the reality is it usually takes days or weeks to get
results back from sequencing services.
Restriction digests give us a way to verify the structure of our
constructs in a single experiment. The first step is to create a
file containing the DNA sequence that we should get if the
experiment worked. The hypothesis we want to test is that the
construct is correct. Computer programs can predict the bands that
should be produced by digestion with various restriction enzymes.
If the plasmid DNA gives the predicted bands, then the hypothesis
is true, and the construct is probably correct. If we get
different bands than those predicted, that hypothesis is false,
and we know that the construct is different from what we intended.
Create a directory containing the test
Inside your PLNT2530
directory, create a sub-directory called as3, to hold materials
associated with Assignment 3. Save all files in your PLNT2530/as3
Copy the following files from your Ugene/Intro directory to your
To make it easier to write
your report, you can download template files in LibreOffice or MS-Word format. These documents
contain dummy data to be replaced with your own data. For
reference, a PDF file is
1. (6 points) Demonstrate the effect of
sequence topology on restriction digests.
critical to realize that the topology of a
sequence ie. whether it is circular or linear,
drastically affects the fragments
predicted in a restriction digest. If the
topology is set incorrectly for a
sequence, the results of a restriction site
search will be wrong. In this section,
we will save the pBS_SK-GUS construct
is a commonly-used sequence format
that contains only the sequence and a
short definition line describing it.
There is no way to specify the
topology of a sequence in a FASTA
file. Consequently, most programs will
consider sequences in this format to
goal is to compare restriction fragments produced by the circular
and the linearized sequences for pBS_SK-GUS. Since we already have
pBS_SK-GUS in its circular form, we next need to create the
In your report, answer the
- Open pBS_SK-GUS.gb
- Select the sequence,
and then choose File --> View sequences. Set
the format to FASTA, and click Run. The FASTA file will appear
on the screen.
- To make it easy to
distinguish this sequence from the original, change the name
of the sequence (found on line 1) to pBS_SK-GUS.fsn.
- Save the file to
your as3 directory as pBS_SK-GUS.fsn.
- Open pBS_SK-GUS.fsn
in your bldna window.
- Run BACHREST
searches on both sequences, and save both output files as pBS_SK-GUS.bachrest
a) What is different about the fragments predicted if we digest a
circular sequence as if it were linear?
b) In the BACHREST output, compare the results for EcoRI and
HindIII, between the linear and circular sequences. Aside from
what you already discussed in a), one of the sites appears
to be missing. Explain the cause.
|Notes on Restriction
1)Remember that the three rightmost columns
in the BACHREST output, with the column headings Frags,
Begin and End, give the sizes of fragments seen in
descending order of size, as they would appear on a gel,
and the beginning and end of each fragment.
2) Some enzymes recognize several possible restriction
sequences. For example, SmlI recognizes 5'C^TYRAG3', where
Y stands for pyrimidine (C or T) and R stands for purine
(A or G). See Sequence
File Formats for a complete list of ambiguity
symbols for nucleotides.
For both parts of this
question, include in your reports examples of BACHREST output for
specific enzymes that support your conclusions. Present the data
in the boxes provided in the template.
2. (6 points) Create a construct using
pBluescript SK(+), for comparison with the construct made
using pBluescript SK(-).
a) Using the same
procedures as detailed in the Introduction
to Ugene tutorial, create a construct by cloning the same 3
kb 35S-GUS fragment from pBI121 between the EcoRI and HindIII
sites in pBluescript SK(+). Call this file pBS_SK+GUS.gb.
b) For each construct, create a map in the Ugene circular
Overview, and export as PNG files to pBS_SK+GUS.png and
Import your maps as shown
in the template.
3. (6 points) Use BACHREST to find
restriction digests that would allow us to determine which of
the two Bluescript vectors was used in the real construct.
As shown in the Bluescript
map, only difference between pBS_SK(+) and pBS_SK(-) is that
the f1 origin of replication is in opposite orientations in the
two vectors. This fragment is in the plus orientation in pBS_SK(+)
and the minus orientation in pBS_SK(-). Because there are several
Bluescript vectors with similar names, unless meticulous lab notes
are kept, it may not have been recorded which of the vectors was
actually used for cloning. Unfortunately, this happens more often
than you might think in the lab.
Fortunately, if we create the sequences for the insert cloned into
both vectors, we can find restriction enzymes that will give
different fragments if the insert was cloned into pBS_SK(+), than
would be seen if it was cloned into pBS_SK(-). Therefore, with a
few restriction digests in the lab, we could find out which vector
was actually used.
Comparison of the digests
should reveal a number of enzymes that give easily distinguishable
banding patterns if each of the real constructs was run a gel. Of
these, choose enzymes for illustrating the differences, using the
- Read pBS_SK+GUS.gb
- Run BACHREST on this
sequence to compare restriction fragments that would be
generated by different enzymes with those generated from
pBS_SK-GUS. From the text editor, save the BACHREST output to
In your report, display
your results in the boxes provided, showing fragments from both
constructs for comparison. You should be able to find at least
three digests that easily distinguish between the two constructs.
- Try to choose
enzymes that don't cut at a large number of sites. On the gel,
it may be difficult to resolve all bands. At the same time,
enzyme cutting sites may appear crowded on the map,
confounding the results.
- Because measurement
of fragment sizes on gels is not very accurate, don't depend
on being able to distinguish fragments whose sizes differ by
- Also assume that
measurement of fragment sizes below 200 bp will be even less
As a second means of comparison, create restriction maps of each
construct in Ugene, showing just the three restriction enzymes you
have chosen, plus EcoRI and HindIII. Export these maps as
pBS_SK+GUSrestmap.png and pBS_SK-GUSrestmap.png. These should be
included in your report as shown in the template.
Discussion: Based the restriction digest data and the maps
of the two constructs, roughly where is the f1 region on your
maps? Explain how your data lead you to that conclusion.
|Note on maps
Where two or more restriction sites are very
close together, the precise ordering of those sites shown
in the map may not be correct, depending on how Ugene
draws the map.
4. Presentation of the
The report should include
In addition to your
report, also upload to the UMLearn Dropbox any new GenBank, FASTA
or BACHREST files created for this assignment. Don't bother
uploading the png image files. These should be embedded in your
- Your name and
student ID number.
- Part 1: Answers to
questions a and b, along with supporting restriction digests.
- Part 2: Insert the
Overview maps of the two constructs (from pBS_SK+GUS.png and
pBS_SK-GUS.png) for comparison
- Part 3: BACHREST
output showing ONLY those enzymes chosen from each construct
that distinguish between the two vectors used. Also,
circular maps from the two constructs, showing the same
enzymes used in the BACHREST.
- Screenshots should
show the data requested, and be readable.
- Screenshots should
only show the relevant window(s), not the entire screen.
- When saving files in
Linux, it is usually best NOT to include blank spaces in file
Use fixed fonts for program output
Many programs generate output that only makes sense with a fixed
font. Most fonts commonly-seen in documents are proportional
fonts, meaning that narrow letters such as 'i' or 'l' take up very
little width, whereas wide letters such as 'O' take up more space.
In fixed fonts, all letter and numbers take up exactly the same
width on a line of text. For example, output from BACHREST,
listing restriction cutting sites and restriction fragments for a
sequence cut with the enzyme AcoI, are shown in both fixed and
proportional fonts. Make sure that when output is shown on a web
page, it is in a fixed font.
Frags Begin End
2817 2533 5349
977 1556 2532
2817 2533 5349
977 1556 2532
Most fonts are
proportional, eg. Helvetica, Times.
Examples of fixed fonts include Courier, Liberation
mono and Terminal.
Your PDF report, along
with associated GenBank, FASTA and BACHREST files, is due by 11:59
pm, Wed. March 21 on the PLNT2530 UMLearn dropbox site in the
Bioinformatics3 folder. Files in word processing formats (.doc,
.docx, .rtf, .odt) are not acceptable.
If you have
questions, it may help to send me a message at