Fourth Homework Assignment
Drawing Restriction Maps
Predicting Gel Electrophoresis Results Based on Maps
Predicting and Interpreting Results of Southern Analyses
Example: chicken genomic digest probed with chicken ovalbumin cDNA fragments
Introduction
The chicken ovalbumin gene was one of the very first genes in a higher eukaryote in
which intervening sequences were found. Workers in the late 1970s and early 1980s used
results from Southern blotting to formulate the hypothesis that the chicken ovalbumin gene
contained intervening sequences. They designed additional Southern blotting experiments to
further test the hypothesis. Finally, when both the cDNA and the genomic DNA had been
sequenced, the investigators were able to confirm their results directly. These exercises include
the Southern analyses the ovalbumin investigators might have done.
Learning Objectives: Analytical skills
*Using annotations provided by GenBank, you will be able to locate important features of
a gene on the sequence.
*Given restriction maps and/or cut site data, you will be able to draw expected agarose
gel results.
*Given a genomic DNA Southern blot of a predicted genomic digest, be able to show
where on the blot you would expect to see signal with a full-length cDNA probe.
*Given a genomic DNA Southern blot of a predicted genomic digest, be able to use
selected probes from within a cDNA to help make an accurate genomic restriction map
or to place the coding regions on a genomic map.
GUIDED ACTIVITY I: Using the computer to map and locate the exons in the chicken
ovalbumin gene.
I.1.
On a separate sheet of paper, draw a linear restriction endonuclease map to scale, using
your results from NEBcutter for the chicken ovalbumin gene (not the cDNA). Use a scale
of 1 cm = 500 bp.
a.
long.
For the chicken ovalbumin mRNA, the line was:
For the gene, the line will be:
1873/500 = ________ cm
_____/ 500 = ________cm
long.
b.
Label the 5’ end of the map “1” for the beginning nucleotide number and label the
3’ end with the appropriate ending nucleotide number.
c.
On the map, use your ruler to position the cut sites for each of the following
enzymes, as appropriate according to your printouts.
Bam HI, EcoRI, HhaI, Hind III, PstI, XbaI, NotI
d.
I.2.
a.
You should have five Eco RI sites on the genomic map, resulting in six
fragments.
Number those six fragments 1-6, from left to right. Remember to number the
fragments, not the cut sites.
Use your computer printout of information and sequence of the ovalbumin gene
from the First Homework Assignment.
i.
On the printed sequence, highlight the exons. Note: the annotation
has a historical error regarding the first two exons. Exon 1 runs
from 1343-1390. Exon 2 runs from 2979 – 3163. The annotations for
the remaining Exons are correct. You should wind up with 8 exons.
ii.
Draw a vertical slash through the sequence to show the transcription
start site. (What nucleotide number is it?)
iii.
Circle the start codon. (What nucleotide numbers form the start codon?)
iv.
Put a square around the stop codon. (What nucleotide numbers form the
stop codon?)
b.
Highlight the regions of exons on the map you drew in #1 above. Be sure to
work to scale.
c.
Using the sequence information, the knowledge of the location of the exons in
the gene, and your results from NEBcutter for both the cDNA and the gene,
i.
Locate the PstI site in the genomic sequence that corresponds to the
PstI site in the cDNA. Draw a box around it in the genomic sequence.
ii.
Locate and draw a box around the Xba I site in the genomic sequence
that corresponds to the Xba I site in the cDNA.
d.
Answer the questions below based on your work so far.
i.
Why are there no EcoRI sites in the cDNA sequence even though there
are several EcoRI sites in the gene sequence?
ii.
Why are there no Hind III sites in the cDNA sequence?
GUIDED ACTIVITY II: Using Southern analysis to demonstrate the existence of introns
and to map genomic restriction fragments. You will not be able to answer this correctly if
you have not understood and completed #I.1 and #I.2 above. You are going to reconstruct
results that investigators in the late 1970s got from gel and blot hybridization analysis.
They did not yet have sequence!
II.1.
Fill in all but the last column of the attached Table, using the map you drew in #1 above,
the cDNA map you drew last time, and the information you have from your marked up
computer printout of the ovalbumin gene sequence. Note that the table is about the
ovalbumin gene, not the cDNA.
II.2.
In Lane 1 of the diagram below, sketch the positions in the gel at which you would expect
EcoRI fragments 1-6 to be located if you had digested complete chicken genomic DNA.
(Remember that if you were actually to run a gel of digested chicken genomic DNA, it
would appear as a continuous smear because there would be so many other fragments
in addition to the ones containing the sequence of the ovalbumin gene.) Remember that if
you had actually digested chicken genomic DNA, you would not know the exact positions
of Fragments 1 and 6, because you do not know the location of the next EcoRI site to
either side of the mapped region. Put each of these two fragments in any logical place.
Reproduce the diagram you made in Lane 1 in Lanes 2-4. At this point, Lanes 1-4 should
look identical. In each lane, as specified below, circle the fragments that would be
positive after hybridization with the cDNA probe mentioned. Use the table you filled out
to help you.
Lane 1 – the full-length cDNA
Lane 2 – the cDNA fragment 5’ of the Pst I site
Lane 3 – the cDNA fragment located between the PstI and Xba I sites
Lane 4 – the cDNA fragment located 3’ of the Xba I site.
1
1
2
3
4
10,000 bp
7,0001
4,000
1,000
250
Analyses of Southern Data
Imagine that you are one of the early investigators of the ovalbumin cDNA and gene. You have
restriction enzyme and gel electrophoretic analysis of ovalbumin cDNA, and you have Southern
blotting data of chicken genomic DNA probed with the ovalbumin cDNA and its fragments, but
you have no sequence yet. In other words, you have only the experimental information from the
gel electrophoresis of the cDNA from the third homework assignment and the Southern blotting
and probing of the genomic DNA as you did it on the previous page.
II.3.
Fill in the last column of the table, numbering the fragments in the order in which you
would expect them to appear in the gel. You will have one or more fragments for which
you can’t be certain of the order. Note any such fragment with a “?”.
II.4.
Explain why the data shown in Lane 1 of the Southern blot and the data from restriction
enzyme digests and gel electrophoretic analysis of the ovalbumin cDNA (last time’s work)
would lead you to make the hypothesis that the ovalbumin gene must contain intervening
sequences.
II.5.
Explain how the results in Lanes 2-4 of the Southern blot further support the hypothesis.
II.6.
Explain in words and with a diagram how your cDNA RE map and your Southern results
would together allow you to partially determine the order of the genomic ovalbumin
restriction digest fragments in the gene without knowing sequence. Note that the answer
to this question requires absolutely no knowledge of sequence!
Table. Analyzing EcoRI digestion and Southern blot analysis of the chicken ovalbumin gene
Fragment
number
1
2
3
4
5
6
Length of
fragment in
base pairs
> 1731
458
486
2362
1806
> 2363
Is the 5’ end
of the
fragment in
an intron?
Is any exon
sequence
present in the
fragment?
(Yes/No)
(Yes/No)
Not
applicable
Southern blot hybridization analysis of total chicken DNA digested with
EcoRI
Try to number
To which of the genomic fragment(s) will each cDNA
the fragments
probe hybridize?
in the order of
(+/-)
their
Full length
Fragment
5’ of the Pst I
site
Fragment
between the
PstI and XbaI
sites
Fragment 3’ of
the Xba I site
appearance in
the gel from
top to bottom.
Use “?” for
unknowns.