D. How many times would you expect to find a specific 20 base pair sequence in the human genome?
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- Part 3. Restriction Enzymes 1) Consider the sequence of DNA given below and answer the following questions 5’ ATTGAGGATCCGTAATGTGTCCTGATCACGCTCCACG 3’ 3’ TAACTCCTAGGCATTACACAGGACTAGTGCGAGGTGC 5’ a) You cut the sequence of DNA shown above using BamHI (see table 19.1 from the text). How many fragments of DNA would you expect to result from this restriction digest? b) If you cut the sequence of DNA shown above using BclI (recognition sequence = 5’ TGATCA 3’, enzyme cuts after the first T) instead of BamHI how many fragments do you expect? 2) For each given sequence/restriction enzyme pair, determine how many pieces of DNA would result form the digest and indicate whether those pieces would have blunt or sticky ends. NOTE: in the given recognition sites, the dash represents where the cut is made. a) HpaI, recognizes 5’ GTT – AAC 3’ 5’ GGATGTTAACAATCTCTACGGGTTAACACCCTTGGGTTAACATCCGCGG 3’ 3’ CCTACAATTGTTAGAGATGCCCAATTGTGGGAACCCAATTGTAGGCGCC 5’ Number of fragments of DNA:…Section A: Linear DNA Common restriction enzymes include: EcoRI, Hindll and BamHl and their sequences are as follows, with the cut site indicated by the arrow (figure 1). Please note that A DNA refers to linear DNA in this tutorial. HindIII 5'..A AGCT.3' 3'....TTCGA A...5' EcoRI 5'..G AATTC...3 BamHI 5'....G GATCC...3' 3' .....CTTAA G..5 3'...CCTAG G...5' Figure 1: Restriction sites of restriction enzymes. When DNA is cut with restriction enzymes, the fragments can be seen on an agarose gel (see figure 2). Base Pairs 21220 25.000 10.000 8,000 6.000 5,000 6557 4361 1641 7233 4,000 3,000 2,500 7421 2.000 5804 E643 1,500 1.000 4878 750 -564 S00 E 3530 125 250 A cut with EcORI A cut with Hindil A cut with BamHI A C D Figure 2: DNA fragments on an agarose gel showing the following: A) a molecular ladder, B) DNA cut with EcoB, C) DNA cut with Hipd, D) DNA cut with Bam The above figure shows the size of each of the fragments/bands produced when A DNA is cut with each of these restriction…Part A Which of the following statements concerning restriction enzymes is true? Select all that apply. ►View Available Hint(s) ☐ Restriction enzymes specifically target and cut RNA in a sequence-specific manner. Restriction enzymes occur naturally in viruses as a defense mechanism against bacteria. Some restriction enzymes generate overhangs in the target DNA sequence upon incubation. During a cloning experiment, the vector and target DNA should be cut with different restriction enzymes to ensure that sticky ends are generated. Submit
- Part 2. PCR 1) In one color, write out the forward primer (5’ GATAC 3’) in the correct position relative to the given template DNA sequence. In a second color, act as the polymerase and fill in the rest of the new strand of DNA Primer/New Strand Template DNA: 3’ TAGCTATGCGGACCTCATGCATTAGAGTAG 5’ Part 3. Restriction Enzymes 1) Consider the sequence of DNA given below and answer the following questions 5’ ATTGAGGATCCGTAATGTGTCCTGATCACGCTCCACG 3’ 3’ TAACTCCTAGGCATTACACAGGACTAGTGCGAGGTGC 5’ a) You cut the sequence of DNA shown above using BamHI (see table 19.1 from the text). How many fragments of DNA would you expect to result from this restriction digest? b) If you cut the sequence of DNA shown above using BclI (recognition sequence = 5’ TGATCA 3’, enzyme cuts after the first T) instead of BamHI how many fragments do you expect? 2) For each given sequence/restriction enzyme pair, determine how many pieces of DNA would result form the digest and…3. A linear DNA fragment is cleaved with one, two or three restriction enzymes to yield fragments as follows: Enzyme(s) Fragments (kb) HindIII 2.5, 5.0 Smal 2.0, 5.5 HindIII + Smal 2.5, 3.0, 2.0 HindIII+ Smal+ EcoRI 1.5, 2.0, 2.5 Draw the restriction map of the original DNA fragment, indicating the positions of all restriction sites.23. Important elements of a directed evolution experiment (“evolution in a test tube”) for an ATP-binding aptamer include: MARK ALL THAT APPLY. Group of answer choices Mutagenic PCR DpnI restriction enzyme Randomized DNA pool ATP affinity column
- you carry out DNA editing using CRISPR whter the editing template DNA has strands labeled with heavy nitrogen 15N. The experiment is carried out in presence ofnormal light isoptope 14 N. The expected distribution of the isotope in the strands in the edited region of the target DNA would be: 1. only one strand is labeled iwth 14 N and the other with 15 N 2. both srrands are labeled with 15 N 3. both strands are labeled with 14 NQ.1. Imagine you are working for a research laboratory and you are asked to help withthe following:a. Cut the following segment of DNA using E. Co R I. Write the sequence offragments clearly and separately. ATTTACGAATTCTTCCAAGAATTCCTAAATGCTTAAGAAGGTTCTTAAGG b. Show sticky ends? How restriction endonuclease are different from the restriction-modification system?Genetic Engineering Process (GEP) # 1: (What kind of process?) Picture A (Sequence #_ DNA introduced into bacterial cells Picture B (Sequence #, DNA ligase added, seals overhangs TTAA AATT AAT AATT TAA TTA TAA PATT PATT AATT recombinant DNA molecules Picture C (Sequence #. donor DNA vector vector and donor DNA digested (cleaved) with restriction enzyme AATT AATT 1477 AATT TTAA overhangs TTAA 1477 Picture D (Sequence #. AATT mixing recombinant DNA molecules replicate and cells divide
- EcoRI recognizes G A-A-T-T-C sequence and cleave/ cut between G and A. How will the DNA fragments look like if EcoRI is used for the DNA below? How many fragments are produced? 5- AAAGATTTGAATTTCGAATTCAATTTAAGAATTCCCTTAGAATTTCC -¹3Im designing a restriction cloning experiment. My professor said that plasmid and insert DNA work best in a 1:1 ratio. If my reaction does not work the first time I perform it, how would I ensure that the cause was the difference in concentration of reagents and how would I fix it so that the I have the correct concentration? If a restriction cloning experiment does not work, how could I be certain that it was due to an incorrect ratio of plasmid:insert and how can i fix this?25. The restriction enzymes Kpnl and Acc651 recognize and cleave the same 6-bp sequence. You have a plasmid and a linear DNA strand that both contain a Kpnl and Acc651 sequence in the same orientation as shown below. You digest both DNA pieces with both enzymes and then attempt to ligate the sticky ends, followed by treatment with DNA ligase. What will happen? 5' GGTACC3' 5' G G T ACC 3' 3' CCATGG 5' y CCATGGS Kpnl Acс651 A) You will produce sticky ends but the two types of ends will not ligate. Instead, you may produce a small amount of religated plasmid where the digested plasmid sequence re-inserts. B) You will produce a recombinant plasmid in which the linear DNA strand is ligated in between the two sites, suitable for cloning. C) You will produce blunt ends that will not ligate because the two restriction enzymes will both operate on both of the sites. D) All the DNA will be completely digested as if you had applied a general DNAse enzyme.