Biotechnology Please hand in separate answers to Q1 and Q2. 1. (...

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Biotechnology Please hand in separate answers to Q1 and Q2. 1. (a) You perform a plasmid prep on a portion of an overnight culture grown up in ampicillin-containing media, cut a portion of the DNA with various restriction enzymes and analyze the resulting bands on an agarose gel. You conclude that the patterns seem to come from one major plasmid contaminated with less intense bands (about 10% as intense on average) from a different plasmid (of similar, but not identical, totalsize). (i) What is the easiest way to derive a pure preparation of the more abundant plasmid? (ii) Might you consider a different approach as possibly being more efficient if you wanted a pure preparation of the less abundant plasmid? (b) You are making genomic DNA preps from a large number of individuals (A, B, C...) and amplifying a specific 2kb fragment (from an autosome, so there might be two variants in a single person) by PCR for sequencing. For one particular sample you are worried that you might have accidentally mixed samples derived from individuals A and B together. When you examine the sequence derived from this amplified DNA you see that most of the sequence is clear but at two positions there are two peaks (position 240 has an estimated 40% C, 60% T peak; position 400 has roughly 30% G, 70% A). (i) You might think that the proportions of the C and G peaks above ought to tell us if the DNA was contaminated (50% more likely if pure, 25% more likely if contaminated). The most obvious variable is that the amounts of A and B DNA (if both are present) might not be equal. What other significant quantitative variable is there that could account for departures from 50% or 25% representation in peak areas? (By the way, although you might think that abnormal bases (mutations or polymorphisms) are generally rare (true) and hence that you can consider certain outcomes more likely than others, in this set of questions and generally with diagnostics you want experimental evidence to prove definitively what genotypes are, rather than making conclusions about the most likely genotypes). (ii) You want to know if DNA that has C at position 240 has a G or an A at position 400. How could you do that with no cloning step? It is possible that the test you make correctly informs you that there are DNAs present that have C-240 and A-400 as well as C-240 and G-400! (iii) Would that result lead you to make a conclusion about contamination? (iv) Would any other result allow you to make a conclusion about contamination? (v) If you are allowed to use a conventional plasmid cloning step prior to any DNA sequencing how (in outline) would you try to determine what DNAs are present in the original sample (assume that it is straightforward to ligate the fragment into an appropriate vector without giving any details)? 1 (vi) If you want to have 500ng of a pure DNA species from the original mixture of DNAs (i.e. to “clone” one type of DNA present) but you are not allowed to use any bacteria (i.e. no conventional cloning), how might you do that? Explain how confident you would be that your final product is indeed pure. (vii) Instead of pursuing any further tests on the potentially mixed DNA samples, you get new samples of DNA from individuals A and B (after seeing the first sequencing results described above). What is the fastest way to determine which nucleotides are present at residues 240 and 400 of each homologous chromosome for each individual (all “haplotypes” present)? Please start Q2 on a new piece of paper 2. This question is about strategies for building cloned DNA “constructs” from cloned components of known sequence. A common objective is to make a cloned DNA that will permit expression of a specific gene when introduced into mammalian cells. The introduced DNA has three functional components- a promoter that drives transcription, the gene (usually in the form of the entire coding region from a cDNA) and a sequence that directs polyadenylation of the transcript made from the cDNA. Here the promoter and polyA-directing sequences are in the same plasmid expression vector for convenient cloning. This plasmid also contains a short coding sequence downstream of the promoter. The encoded amino acid sequence forms an epitope that can be bound by a widely available antibody (a “Flag tag epitope”). The objective is put the appropriate portion of cDNA coding region downstream of this tag sequence and upstream of the polyadenylation signals in the expression plasmid. The product would be known as a “translational fusion” because translation of protein from the RNA transcribed from the plasmid would initiate at the AUG of the Flag epitope. Translation would then continue in the same reading frame into the cDNA sequence and hopefully produce a fusion protein with Flag at the N-terminus followed by the protein encoded by the cDNA. Here, you have both component plasmids (diagrams below; sequences are written with spaces between triplets to make it easy to see relevant reading frames) purified, in plentiful amounts and with entirely known DNA sequences. For all restriction sites shown there is no additional unmarked site (and we will work only with the sites that are shown). Enzymes cut at the location indicated within recognition sequence on each strand (all written 5’ to 3’). BamHI G^GATCC BglII A^GATCT XbaI T^CTAGA NcoI C^CATGG KpnI GGTAC^C 2 (a) Imagine that your first plan is to ligate the BglII-XbaI cDNA fragment between the BamHI and XbaI sites of the vector (note that BamHI and BglII give complementary sticky ends and that inclusion of the 3’UTR region of the cDNA in the product is fine). I suggest that it is worthwhile purifying the 2.3kb BglII-XbaI fragment and the 5kb BamHI-XbaI fragments on an agarose gel after cutting but before ligation. The purpose of these purifications is to make sure you get enough of the desired product and reduce the amount of competing products you would need to screen through at the end of the cloning experiment. (i) What would likely be the most problematic competing products AND how serious a problem would these be if you did not purify the two fragments to be ligated? 3 After incubating the two purified fragments with T4 DNA ligase under suitable conditions (ii) how many different DNA products will there likely be? (iii) what proportion of all of the DNA will be the circular product that you want? (iv) should you purify the desired product on a gel before transformation? Your objective is to have successfully made a cloned DNA with the BglII-XbaI cDNA fragment incorporated into the expression vector. (v) What are your next steps to COMPLETE this process (be specific: imagine you are outlining to your lab supervisor what you will do in order to be able to hand over a tube containing the requisite DNA within 2-3 days)? (vi) The product you are trying to make this way will not, in fact, have the potential to express the desired Flag-tagged fusion protein. Explain why not by writing out relevant sequences clearly. You consider alternative ways of making a very similar product that can encode the desired fusion protein. In all cases (unless otherwise stated) the encoded fusion protein must have all of the written amino acid residues of the Flag tag and should include the entire coding region of the cDNA but it can have anywhere between zero and ten codons (encoding any amino acids) between the two. (b) You consider using an adapter (made from hybridizing two oligonucleotides together- theoretically any sequence you chose, any length). You first consider an adapter that could be ligated between the BamHI and BglII ends of the two fragments. (i) Write out a possible adapter sequence that would work (make clear why it would work). (ii) What strategy would you use to try and maximize the proportion of correct ligation products at the end of your procedure compared to ligation products that would entail significantly more screening work? This is an unusually challenging task, so it is reasonable to consider a multi-step procedure and all available tools. It is also worth stating limitations. Because there are some potential difficulties with the strategy above you also consider using the KpnI site instead of the BglII site to generate a cDNA fragment. (iii) Write down a suitable adapter sequence for this (show adjacent sequences to be ligated and relevant reading frames to illustrate why your approach should work). (iv) Could you also, if you wish, connect the KpnI site to the NcoI site of the expression vector with an adapter? (v) Given the above considerations (and any suitable extrapolations) can you summarize what disposition of unique restriction sites (sites that occur more than once in the expression plasmid or in the cDNA are usually very hard to incorporate into good strategies) is required in an expression vector plasmid and a cDNA plasmid in order to make a fusion protein expression construct using a ligation strategy that employs an adapter? Try to be precise in your language and give any relevant numerical estimates. (c) You also consider using PCR as part of your strategy to make the desired clone (any suitable construct that will encode a correct fusion protein). 4 (i) If you decide you will still use the BamHI-XbaI expression vector fragment describe how you would generate a suitable cDNA partner fragment for ligation. You must pay attention to both ends but you need to write out sequences (and describe steps accurately) to show exactly the PCR primer sequence AND (noting the intermediate steps) how a correct junction will be made for the end that connects to the expression vector BamHI site. (ii) If the task were modified such that the final product must encode EXACTLY the amino acid sequence MDYKDDDDKMVYSGTV... how would you do that? (d) You may correctly consider that choosing between a good adapter strategy and a good PCR strategy for the cDNA in this example is not easy. Both involve making some synthetic oligos, some gel purification and ligations. Nevertheless, if you were commonly going to perform this type of experiment (say for a dozen different cDNAs over a few months) I think you would choose to use a PCR strategy. Why? 5

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1. (i) Answer: When the different DNA fragments, get inserted into plasmid cloning vectors, they yield a mixture of recombinant plasmids, containing a single DNA fragment. These recombinant cells are then treated with CaCl2 and further incubated with the blend of recombinant plasmids and are then plated on nutrient agar (NA) containing antibiotic ampicillin. Every colony of transformed, antibiotic-resistant cells which grows arises from a single cell that takes up one or another form of the recombinant plasmids; thus all the cells in a given colony carry the same DNA fragment. Visible colonies are obtained after overnight incubation of cells at 370C. Copies of the DNA fragments are isolated from the new mixtures as the colonies are separately growing on different culture plates. Thus we can derive more abundant transformed cells contain multiple copies of a given plasmid using the above protocol.

(ii) Answer: The clone library is enriched with a number of clones covering a large part of diversity. To screen less abundant plasmids, one can go for subtractive hybridization protocol which has been designed to screen subdominant members in a clone library. Another approach is to repeat the PCR and clone library construction after phylogenic analysis on the dominant colony. Peptide nucleic acid probes are designed specifically for dominant clones, and PCR reaction is preceded. This PCR camping is a preferential method for less abundant sequences from mixed templates...

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