Biotechnology
Homework 1 Fall 2010
Hand in on Wed 9/22
Please write clearly and leave plenty of space for comments. Be concise but precise in your
answers and, by all means, add questions about what you do not understand. Then we can
clarify these issues. Points available are indicated in [ ]. Read the questions carefully- they aim
to be specific and to give you some guidance about what you need in your answer. You will
always need to explain your reasoning (and perhaps more carefully than you are used to doing),
even if the question does not say “Explain”.
PLEASE START A NEW PIECE OF PAPER IN YOUR ANSWER WHERE REQUESTED.
We split up the grading by questions and therefore need to separate your homework into sections
distributed to separate graders.
1. DNA Hybridization in solution.
In the absence of extensive practical experience, it is hard to develop a feeling for the specificity and
extent of DNA hybridization and hence whether a designed experimental approach is practical or not.
Formulae can help.
One commonly used approximation is that Tm = 2 x (number of A-T bps) + 4 x (number of C-G bps)
for a DNA oligonucleotide of up to 13 nt hybridizing to a complementary target.
Tm (melting temperature) is when 50% of double-stranded DNA is dissociated into single strands.
For longer oligonucleotides this becomes a bit more complicated:
Tm = 64.9 + 41 (# of CG bps – 16.4) / Length of oligo
In each case, the answer is in degrees centigrade (C), it is only an approximation (more accurate
calculations must take into account the precise sequence) and the equations assume conditions of
50nM oligonucleotide, pH 7.0 and 50mM salt (NaCl). These equations can be used to make rough
calculations about oligos hybridizing to DNA on a solid support (Southerns, microarrays etc.) or in
solution (for PCR, DNA sequencing etc.).
(i) Why do you think 50mM salt (at least as an approximate concentration) was chosen for quoting
(designing) these formulae?
[1]
If the salt concentration is different the Tm must be adjusted by adding a factor equal to
16.6 (log10 [Na+]/0.05) where [Na+] is the molar salt concentration
(ii) By how much is the Tm raised if you increase the salt concentration from 50mM to 0.5M?
[1]
(iii) If you are conducting a hybridization experiment and you want more correctly hybridized product
what are TWO of the most important factors that you can change (excluding salt concentration)?
Explain carefully (indicate directions of changes and whether there is an optimum).
[2]
Mismatches between two hybridizing DNA sequences reduce the Tm by up to about 5C per mismatch
for oligos (and by about 1C per % mismatch for long DNAs).
(iv) Imagine an application where you would normally use an oligo of 20nt that matches its target
perfectly and you have adjusted hybridization conditions optimally. Now you want to use a different
oligo in the same type of experiment (where you can assume that the key issue is hybridization of the
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oligo to its single desired target site) but the oligonucleotide must have FOUR mismatches with its
intended target. Consequently, you choose to increase the length of the oligonucleotide (and you can
change hybridization conditions if you wish).
What length of oligonucleotide (and changes, if any, in hybridization conditions) would you choose?
You should think about the objective of the experiment and not just use a formula without thinking of
the context. If it is simpler for you, or important, you can describe the application that you are thinking
about as an example.
[2]
(v) Several restriction enzymes generate 4nt single stranded overhangs (e.g. 5’ AGCT 3’ for HindIII).
Using the formulae given above it is clear that at low enough temperature or high enough salt
complementary four nucleotide overhangs could be reasonably stable. You might think, for example
that you could have high enough salt and low enough temperature (well above freezing) that, from the
simple formulae given above, would lead to less than 50% of 4pb duplexes dissociating into single
strands at equilibrium. However, as you know, if a DNA molecule (say a plasmid) is being
constructed for transformation into bacteria, one always uses T4 DNA ligase to join two
complementary HindIII-cut fragments together. Ligation obviously improves efficiency but why does
it make such a big difference that it is effectively essential for such cloning experiments?
[1]
(vi) Do you think that T4 DNA ligase would be essential for joining two molecules together prior to
bacterial transformation (as above) if complementary single-stranded overhangs were considerably
longer than 4nt? Explain carefully, thinking about the whole process and being clear about what you
do not know (and hence where you are making an informed guess).
[1]
(vii) Restriction enzymes very conveniently produce complementary single-stranded overhangs,
allowing joining of those ends by ligation. That raises the question of whether there are other ways of
generating single-stranded overhangs of predictable sequence. Using PCR (as I will emphasize later in
the course) you can add any sequence you wish to the ends of DNA fragments but the added sequence
is essentially double-stranded after PCR (the exception being an extra A residue added by Taq
polymerase). How could you generate a 12nt single-stranded DNA overhang of prescribed sequence
at the end of a normal double-stranded PCR product (after the PCR reaction)?
[1]
2. Hybridization to immobilized DNA
It is often crucial to consider the sensitivity of a detection method and instructive to think about how
many molecules you are trying to detect.
Imagine performing a genomic Southern blot using radioactively labeled probes (which used to be
common)
(i) If you were using a 2kb region of DNA as a probe how might you make the probe?
[1]
(ii) If you could make an RNA probe to the same sequences to equal specific activity and in the same
amounts, would it be as effective as the DNA probe? Explain.
[1]
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(iii) How would you make such an RNA probe? Remember that a good answer will always name all
of the key ingredients (as if you were going to do this yourself; except details of concentration, time,
temperature and, in this case, purification are not necessary).
[1]
(iv) If you used genomic DNA from baker’s yeast and a yeast DNA probe would you get a stronger
signal (if so, by how much (a quantitative estimate)) than if you performed an analogous Southern
blot with human DNA (using 5g of digested DNA in each case) and a human probe (made to the
same specific activity as the yeast probe and used at the same concentration)? Explain.
[1]
(v) Even if the human genomic DNA Southern blot described above employs differently labeled
probes it would be quite difficult to obtain a strong signal using an amount less than 0.1 g. Roughly
how many DNA molecules containing the 1kb segment are in 0.1g of human genomic DNA? [1]
(vi) Given the stated sensitivity of a Southern blot in (v) above, how is it possible (by comparison) to
obtain a strong signal in a FISH experiment? Please note that the same labeling and detection
techniques (fluorescence) could be used in each application, so use of different labeling/detection
techniques is not the answer being sought here.
[1]
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PLEASE START A NEW PAGE (SHEET) FOR QUESTION 3
so we can separate 1&2 from 3&4
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3. DNA sequencing
(i) Imagine you have a good preparation of genomic DNA. You want to see if you could use that as a
template to generate a product primed by a single oligonucleotide. You make the oligonucleotide
correctly to be complementary to one site in the genomic DNA, you denature the genomic DNA at
high temperature, add plenty (vast molar excess) of the oligonucleotide, allow high stringency
hybridization at a suitable temperature, add dNTPs (with one radioactively labeled), suitable buffer and
DNA polymerase. After suitable incubation you pass the products through a molecular exclusion spin
column that retains labeled nucleotide but allows DNA (even as short as an oligo) to pass through.
You are pleased to see that most of the radioactive label is in the DNA fraction. However, you also
performed a control experiment in which all steps were identical except you added no oligonucleotide.
Here you also find that most radioactive label is in the DNA fraction. What is the most likely
explanation?
[1]
DID YOU START A NEW SHEET OF PAPER? THANK YOU
(ii) If you tried to perform standard Sanger dideoxy cycle sequencing with a good primer but using
total genomic DNA as template you might very well have a problem with both signal and noise.
Given the observation cited in (i) above, how might you change the standard cycle sequencing set up to
reduce the background?
[1]
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(iii) If you had a normal purified DNA template to sequence (for example, in a plasmid vector of your
choosing, or, if you prefer as a PCR product with end sequences of your choosing) could you in
principle obtain DNA sequence information by using RNA polymerase, rNTPs and rNTPs lacking the
3’-OH as in standard dideoxy sequencing? Explain your reasoning AND if you think that the
experiment could work in principle, why it may be less effective than standard DNA sequencing. [1]
(iv) If you had a pure RNA molecule (a single species in large amounts, analogous to cloned DNA or
a PCR product) could that, in principle, be sequenced directly (converting to DNA and then
sequencing the DNA product would not be considered direct)? How (name ALL key reagents)? [2]
4. Synthetic oligonucleotides & applications
In creating synthetic bacterial genomes of 400-1,000 kb a step-wise procedure of assembling
increasingly large segments was used. In the first set of steps pieces of about 5-7kb were put together
by one of a handful of companies that offer such a service. The exact details of how this is
accomplished vary and are sometimes proprietary but we can look at the principles and options here.
(i) The initial chemically synthesized oligonucleotides could quite easily be made in the range of
roughly 80-200nt (perhaps even longer) in reasonable yields and purity. The choice of what is optimal
depends on synthesis quality and the downstream process but let’s assume here that 200nt oligos are
made. How could these be assembled into a 5kb unit?
[1]
(ii) What do you think would be the most effective way of purifying assembled 5kb units? There will
be many downstream steps in assembling the whole genome so a large amount of pure 5kb fragments
may be needed. In fact, you would be best advised to think about how you might generate 1 g or
more of product even if you only initially make 100ng (no typo)!
[1]
A DNA polymerase generally has the choice of extending a primer annealed to a template or
hydrolyzing the last primer nucleotide using 3’ to 5’ exonuclease activity. Exonuclease activity is
much higher for a mismatched 3’ nucleotide and polymerase activity is much higher if base-pairing is
correct. The competition between polymerase and exonuclease is also influenced by dNTP
concentration and is very different for different polymerases. In general, a polymerase that is fast and
processive in DNA synthesis has low 3’ to 5’ exonuclease activity. Such enzymes are good for DNA
sequencing, including Taq, which is widely used for cycle DNA sequencing.
(iii) Some companies offer oligonucleotides that are either PCR-grade (meaning that you just need to
amplify a product and may not need the product to have exactly correct sequence at its ends) or
sequencing grade. What do you think is likely to be the single most critical difference between these
two grades? Be specific and explain the consequences for DNA sequencing.
[1]
(iv) If a major error were made in oligonucleotide synthesis (very rare), would you be able to use the
product as a primer for DNA sequencing if (a) the first CPG-attached nucleotide was wrong, (b) the
tenth nucleotide was incorrect, (c) the tenth nucleotide was missing, (d) the last (20th) synthesized
nucleotide was coupled to only 50% of the product? Explain your reasoning.
[2]
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