BT 222 Biotechnology Assignment 1 Q1-(10 points) some would like to claim breeding is biotechnology as indirect or in deliberate transformation.

In that way, they can claim that we actually have been using biotech products for a long time. This is a hollow argument that will backfire if pressed? How you could argue with this argument? 1 page with examples. Breeding is a slow process and is imprecise the beneficial gene added is from the same species. The beneficial gene is delivered by mating within the species, and usually large sets of genes of unknown function are also transferred between related organisms. Breeding only exchanges genes found in the species Breeding can transfer the transgene to other breeding materials but it is not the same as biotechnology Transformation is a biotechnology technique and is faster and precise process than breeding. The beneficial gene is added from another species, which is delivered by plant genetic engineering/recombinant DNA technology. Transformation usually involve moving a single gene whose function is known. Biotechnology adds traits not available in the species. Eg: Soybean does not have a gene to breakdown RoundupThe gene comes from bacteria Q2. (20 points) you found a new gene in mice that when mutated causes a disease and so you can produce a disease genetic model mice for studying this disorder. First your plan is to amplify the normal gene and the mutated gene in both normal and the mutated gene in the genetically affected model mice you created using polymerase chain reaction (PCR) and then clone it for sequencing to be sure that the mutation you did in this gene is the direct cause for the disease. a) Based on the size of this gene, you chose the appropriate cloning vector that can carry this gene and you would like first to generate a restriction map of this cloning vector. You cut your vector with various combinations of restriction enzymes and determine the size of the resulting bands by gel electrophoresis. The size of the DNA fragments following the digestion are shown in below table (kb = kilo base pairs; 1kb= 1000bp).

i) What is the length of your vector (in kb)? (2 points) = 15 kb ii) Draw a map of the circular plasmid vector including all restriction enzyme sites and the distance (in kb) between them. (5 points)

You now wish to amplify the normal gene by PCR to obtain enough DNA so later you use it for sequencing. The nucleotide sequences flanking the gene are shown below

b) Which pairs of primers that you could use to amplify the above gene (2 points). #1: 5 GACGCA3 and 5CGCATG3 #2: 5 CTGCGT3 and 5CATGCG3 #3: 5 GACGCA3 and 5GTACGC3 #4: 5 GACGCA3 and 5CATGCG3 c) You design the PCR reaction such that it cycles from 95oC for 1 minute to 52oC for 1 minute, then to 70oC for 3 minutes and then it returns to 95oC. You repeat the cycles 30 times. i) in 4 lines, briefly explain what happens to the DNA at each temperature during one PCR cycle. (3 points) = During 95oC for 1 minute the DNA is denatured, the strands are separated. During 52oC forward and reverse primers start annealing with the original strands of the DNA. And at 72oC the primers are extended by DNA polymerase in the 5 to 3 direction. ii) Suppose you started with a single molecule of DNA, approximately how many molecules of DNA would you obtain at the end of the 30 cycles of PCR reaction? (2 points) = Molecules of DNA = 2n, where n is no. of cycles Molecules of DNA = 230 = d) Why the frequency at which errors occur during DNA amplification through PCR is much higher than that observed during DNA replication? Think about the activity/type of the enzyme used in PCR compared to that one used in DNA replication. (2 points) = The Taq DNA polymerase used for PCR is different from the regular DNA polymerase and doesnt have the proofreading ability which causes higher frequency of errors in PCR. e) You then isolated your PCR amplified gene/DNA, digest it with EcoRI, then when you purified it using gel electrophoresis you obtained a PCR DNA fragment with EcoRI overhangs of 4kb. You then also cut your plasmid vector you picked in part a, with the same enzyme EcoRI, and purify the products fragment/s. (2 points) i. How many fragments you expect to get in your gell after digesting your vector with EcoRI ?

= 2 fragments ii. What is the size of the fragments you expect to obtain when you cut your vector with EcoRI? = 13 kb and 2 kb iii. Which of these fragments would you use for cloning your gene (digested product that purified from the gel electrophoresis)? = 13 kb fragment If you want to combine/ligate the desired vector fragment with the PCR/gene fragment, what enzyme you need to use? DNA ligase After that you transformed E.coli competent cells with the ligation mix and selected for the transformed colonies. Then you isolated these two colonies, and in order to confirm that these colonies have your cloned gene, you Purified your recombinant vector plasmids from each colony, Analyze the plasmid DNA from each colony by digesting the purified plasmid DNA vectors from each colony with EcoRI and analyze the products of this restriction for each digest by gel electrophoresis. Lets say after gel electrophoresis, you observe two distinct (different) EcoRI restriction digestion patterns from the 2 different colonies as shown below.

f) How you explain the observed restriction pattern of purified plasmids #1from colony 1 and purified plasmid #2 from colony 2 as seen on gel diagram. (2 points) = Plasmid #1 is the self-ligated 13 kb plasmid vector that doesnt have the 4kb PCRamplified DNA insert. Plasmid #2 contains the 4kb PCRamplified DNA insert, but does not contain the 2kb EcoR1 plasmid DNA fragment which was removed when the plasmid was cut with EcoR1.