1. Look at the cut DNA sequences shown below in i) and ii). a) What restriction enzyme(s) was/wereused to create these sequences? b) Could the sequences below be hybridized and ligated? c) If it ispossible to join these two sequences by hybridization and ligation what would happen if the resultingsequence was again exposed to the original restriction enzyme(s) that you identified? You mustdefend any answers that you make with logic at the level of course content.i.5'-G3'-CCTAGii.GATCT-3'A-5'

To help you better understand the answers above, here are non-technical explanations of each…

Answered: 1. Look at the cut DNA sequences shown…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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DNA is extracted from the blood cells of two different humans, individuals 1 and 2. In separate experiments, the DNA from each individual is cleaved by restriction endonucleases A, B, and C, and the fragments are separated by electrophoresis. A hypothetical map of a 10,000 bp (base pair) segment of a human chromosome is shown (1 kbp= 1,000 bp). Individual 2 has point mutations that eliminate restriction recognition sites B* and C*. After the 10kbp segment is digested with each restriction endonuclease A, B, and C one by one, samples are loaded onto an agarose gel for electrophoresis for analysis. I would like you to draw the result of gel electrophoresis. To answer the question, first, draw the whole gel image (on the right) on your answer sheet, then indicate where you expect to see the bands on the gel. (Hint1: Same-size bands will appear at the same position. Hint 2: On the gel bands from individuals 1 and 2 might be at different positions.) The left lane (with an M label on the…
1) Prepare the following enzymatic reaction, present it in tabulated form. In a final volume of 30 ul, where buffer 4 (10 ml). How much volume of each reagent would be used and how much of water? Is there any problem? 2) The DNA pol 1 enzyme comes at a concentration of 50,000 U/ml. You have to prepare a 50 ug PCR reaction where you must use 0.05 U/ml reaction. You add 10 ul of PCR buffer, 2 ng of tempered DNA that is at a concentration of 0.5 ng/ul, primers (which are at 200 mM) so that each one remains at a concentration of 200 uM, Mg+2 that is 5 mM (10 X), enzyme and water. Present the table of all the reagents included in the reaction, the volumes of each one in ul. Present where the initial and final concentration of each reagent applies. Assume you have micropipettes for all values.
After mutagenesis of wild type Vibrio fisheri, you isolate two different mutant strains (A and B) that, unlike the wild type cells, fail to luminesce when grown to high density in a flask with appropriate medium. Curiously, however, when you inoculate both mutant strains in the same flask, you observe that the mixed (A+B) culture begins to emit light after growing dense. a) What gene/functions are likely affected in each of the two mutants? b) How does this explain their phenotypes?
For a restriction enzyme that recognizes the restriction site GGCC, Which of the following statements is/are true?
Describe the process for shotgun sequencing of a genome. Practice aligning the two sets of sequenced fragments below, to determine the order of the fragments and the complete sequence.
The optimal design of primers is critical to the effective amplification of DNA sequences. i) Outline the criteria for optimal primer design. i) Illustrate forward and reverse primer sequences using an example of a hypothetical DNA fragment. Give a brief overview of an online primer design software program.
During PCR amplification in preparation for DNA sequencing, why were there different colors at the 3’ ends of the fragments produced? (What did these four colors represent?)
After digesting the DNA produced by PCR (generated using the primers mentioned in question 2 added for context below) and the pET-28a vector with restriction enzymes SacI and HindIII, what steps would be needed to obtain a pure culture of E. coli cells that contain the pET-28a-OXA-M290 plasmid (i.e., a culture which consists only of E. coli cells that contain this plasmid)? And, how could you confirm that the OXA-M290 gene was successfully cloned into the pET-28a vector without any mutations? [4 – 8 sentences suggested] Question 2 (Do not answer, just for context):The forward primer that will be designed is the primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located. In order to clone the PCR product generated using these primers into pET-28a, extra nucleotides must be added to the 5' ends of the primers. These extra nucleotides will contain the…
A 2.0kb bacterial plasmid ‘BS1030’ is digested with the restriction endonuclease Sau3A; the plasmid map is depicted in the diagram below and the Sau3A (S) restriction sites are indicated. Which of the following DNA fragments do you expect to see on an agarose gel when you run Sau3A-digested plasmid ‘BS1030’ DNA? a. 250 bp, 450 bp, 550 bp, 1.1 kb, 1.5 kb and 2.0 kb b. 2.0kb c. 250 bp, 400 bp, 450 bp, 500 bp and 550 bp d. 100 bp, 200 bp, 250 bp, 400 bp, 500 bp and 550 bp
Using the “target DNA” sequence provided in the attached image, what sizes of inserts can be prepared using HindIII digestion? Which one contains the coding sequence?
Why would you NOT expect a restriction endonuclease to exist that would recognize the site AAGGAA?
Transcriptome analysis involves two separate methodologies: gene expression and RNA seq analyses. The 10 items below are a scrambled listing of the steps used in the two procedures. Identify the steps involved in RNA seq from the list below. Use the numbers in the list to refer to each step. Once the steps for RNA seq have been identified, write the steps in the order in which they are performed during the experiment. (1) DNA sequencing (2) Allow for hybridization and wash excess cRNA. (3) Mix labeled cRNA with array chip. (4) PCR amplification (5) Measure fluorescence intensity to determine abundance of transcripts. (6) Add labeled cRNA at each microarray location. (7) Map cDNA sequences to the genome of the organism to determine identity and abundance of transcripts. (8) mRNA isolation from cells (9) Prepare fluorescently labeled cRNA probes (10) cDNA synthesis

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