Biotechnology
Homework 3 Fall 2010
Hand in on 10/6
Please read questions carefully. You always need to provide some explanations of your
answers.
Vectors for cloning large DNA segments
1. I described making genomic libraries with partial Sau3A digests for both lambda phage
replacement vectors (insert sizes generally 20-25kb) and for BAC vectors (inserts generally in
150-300kb range). Imagine the advice to make a library using Sau3A was ignored and that XhoI
(cuts at CTCGAG after the first C on each strand) was used instead of Sau3A for partial digestion
of the genomic DNA (of course the cloning sites in the vector would also be changed
accordingly).
(i) Why is the choice of XhoI worse?
[1]
(ii) Is the choice to use XhoI equally problematic for lambda and BAC libraries or is it a more
serious problem for one type of library?
[1]
(iii) What are THREE changes you could make to the most basic library cloning protocol (of
simply ligating the partial XhoI genomic DNA digest to XhoI cut vector) in order to minimize the
possibility that clones in your library will include composite insert fragments (two or more insert
fragments ligated to each other). You must start with a partial XhoI genomic digest but you can
use whatever lambda vector you find convenient (i.e. invent whatever restriction sites you find
most useful). Be sure to explain clearly what your extra or different steps are (number the
possibilities so it is clear which are distinct) AND why they help.
[3]
2. Imagine you have a partial Sau3A digest library of mouse genomic DNA in lambda phage and
that you pick one clone. You could digest that cloned DNA with several restriction enzymes, run
the products on agarose gels and figure out a restriction map of the cloned recombinant DNA.
You could even sequence the entire cloned DNA (by methods we will discuss shortly). That
would tell you (provided cloning introduced no artifacts) the distribution of restriction enzyme
sites (or DNA sequence) of a segment of roughly 20kb in the mouse genome.
(i) Now consider that you wish to know the restriction map for a 40-50kb segment of the mouse
genome centered on the 20kb region described above. Starting with the first clone and using your
genomic library how might you proceed?
[1]
(ii) If you succeed in the above objective you will have a map and you will probably have more
cloned segments of DNA. Imagine you also have a cloned cDNA for a gene that maps exactly to
the region under consideration.
(a) How could you use the cDNA (without determining DNA sequence) to determine a minimum
size of the gene in mouse genomic DNA?
[1]
(b) Why would that only be a measure, or estimate, of the minimum size of the gene?
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[1]
(iii) Let’s say that you think the gene occupies 30kb and you want to capture the entire gene on
one cloned fragment of DNA. One solution would be to make a BAC library but before BACs
were used as vectors another reasonable suggestion would have been to use a cosmid. I’m sure
you can look cosmids up and find that they have Cos sites, they include a ColE1 plasmid origin
of replication, an antibiotic resistance gene and the vectors are small compared to lambda DNA
because they do not encode any lambda phage genes encoding proteins. Cosmid vectors can be
used to clone segments of around 40kb.
Explain how a cosmid vector is used AND explain what advantage it has over conventional
cloning into a ColE1 type plasmid? If you cannot see a clear advantage it may be helpful to state
clearly what is NOT an advantage and to explain why you have doubts over your best guess for
the advantage offered.
[2]
(iv) Why does a YAC vector include a ColE1 plasmid origin of DNA replication?
[1]
(v) When making a YAC library what do you do after ligating vector and insert fragments
together (to end up with recombinant YAC clones)?
[1]
PLEASE START A NEW PAGE
3. In the “Gateway” cloning system, which exploits sequence-specific homologous
recombination catalyzed by purified proteins in vitro, it is usual to first create an “Entry clone”
and then to transfer the cloned segment to a “Destination vector”.
(i) An Entry clone can be made by recombination of a PCR product with a Donor vector (which
has attP1 and attP2 sites) but how must the PCR product be made?
[1]
(ii) Imagine you made an Entry vector carrying a certain DNA and then successfully transferred
the DNA to a specific destination vector (which has attR1 and attR2 sites). You then lost the
recombinant Entry vector clone and wondered if you could make it again starting from the
recombinant destination vector clone you had made. Can that be done easily? How (you may use
DNAs that are potentially available but not explicitly mentioned in this question)?
[1]
(iii) Imagine you have constructed a cDNA library in a lambda vector by methylating internal
EcoRI sites of double-stranded cDNA fragments prior to adding EcoRI linkers, and then ligating
EcoRI-cut inserts to EcoRI-cut vector. The inserts will be many (say one million separate clones)
and will have different sizes (mostly in the 1 to 7kb range). You have just packaged the ligated
DNAs into phage particles (and everything is going just fine).
(a) You want to send the library to five friends and to keep some for yourself. As far as possible
you want to send (& keep) the complete library. What do you do (explain the actions not just a
summary phrase)?
[1]
(b) You decide that you want to transfer the whole library (as best possible) to a plasmid vector in
the most economical way possible. How might you do that (remembering that you do not want to
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cut cDNA fragments at internal EcoRI sites [or at any other internal restriction site]?
[2]
4.
Using the Quik-change site-directed mutagenesis method,
(i) is DNA synthesized in one round of DNA replication ever used as template in a subsequent
round (as in PCR)? Explain.
[1]
(ii) DpnI is used to digest hemi-methylated DNA and fully methylated DNA at GATC sites.
What does this accomplish (be precise)?
[1]
(iii) What are the molecules that give you the desired mutant clones AND when (precisely)
during the mutagenesis procedure are they formed?
[1]
(iv) Could you use the procedure to remove a precise segment of 30bp from a recombinant
plasmid clone rather than to introduce base substitutions? How?
[1]
5.
(i) If you were using the SLIC method to insert a fragment of DNA into a plasmid vector, and you
were using PCR to generate suitable vector and insert fragments (to be followed by T4 DNA
polymerase exonuclease treatment, annealing and transformation), how would you design the
PCR primers?
[1]
(ii) There are many ways to connect two fragments together as part of a cloning procedure. If we
consider “easy”, “seamless” and “universal” as three potentially important qualities (but only
relevant to some, not all, cloning procedures) how do you rate (a) ligation using naturally
occurring restriction sites, (b) Gateway cloning (for example, after PCR to make an ENTRY
clone), (c) PCR cloning by adding restriction enzyme sites for ligation, (d) PCR followed by
SLIC? Your comments on “easy” will be taken as subjective (& will not be evaluated) but for
“seamless” and “universal” you should explain your ratings simply and briefly.
[2]
(ii) Could you use SLIC to perform site-directed mutagenesis at two positions in a recombinant
plasmid simultaneously? Explain.
[1]
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