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Biology 1202B Lecture Notes - Lecture 16: Dna Ligase, Sticky And Blunt Ends, Restriction Enzyme

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taupellama691
20 Apr 2013
School
Western University
Department
Biology
Course
Biology 1202B
Professor
Brenda Murphy

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Document Summary

Biotechnology technology applied to biological systems or living organisms to make or modify products or processes for a specific purpose. Re recognize short amount of dna sequence (4-8nt long) called restriction sites and cut the dna at specific locations within those sequences with a restriction enzyme to produce restriction fragments. Restriction sites are often palindromes same sequence 5" to 3" from both strands and produces sticky ends: ex. Sticky end can form hydrogen bonds with complimentary sticky ends on any other dna molecules cut with the same enzyme. Complete digestion conditions are such that if a restriction enzyme site is present in the dsdna, the enzyme will cut at all the dsdna sites. Incomplete digestion conditions are such that if a restriction enzyme site is present in the dsdna, the enzyme may cut at all the dsdna sites: conditions include things like temperature, amount of time etc Each human cell has two copies of most genes.

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Related Questions

Recombinant DNA Technology

The questions all relate to the same topic and are somewhat interrelated

1. For gene cloning, a geneticist digests DNA with ___ an enzyme that cleaves DNA at sequence-specific sites.

A. DNA polymerase.

B. Ligase.

C. Restriction endonuclease.

D. cDNA.

2. Certain endonucleases cut DNA and leave DNA termini without overhangs which are called ___

A. Cohesive termini.

B. Sticky ends.

C. Blunt ends.

D. Oligonucleotides.

3. An enzyme found in retroviruses (like HIV) that can be used to prepare DNA fragments from mRNA is called ___.

A. Reverse transcriptase.

B. RNA polymerase.

C. DNA polymerase.

D. Integrase.

4. After combining DNA fragments in a cloning experiment, ___ is used to covalently to join the DNA segments.

A. Restriction endonuclease.

B. DNA ligase.

C. DNA polymerase.

D. Helicase.

a. Fred and Helen’s conceived child: Child ____

b. George and Helen’s conceived child: Child ____

c. George and Helen’s adopted child: Child ____

We have four DNA sequences that we need to digest (cut up), and have 6 restriction enzymes in stock in our lab. We want to find out which restriction enzyme we can use on each sample.

For each sequence, please indicate which restriction enzyme(s) would cut within each sequence. Each sequence may be cut by one, more than one, or no restriction enzymes.

Following the sequences are two questions. Please choose from the same list of enzymes to indicate the correct answer to these questions.

Restriction enzyme Digestion pattern
EcoRI

5' G|AATTC 3'

3' CTTAA|G 5'
HindIII

5' A|AGCTT 3'

3' TTCGA|A 5'
BamHI

5' G|GATCC 3'

3' CCTAG|G 5
AluI

5' AG|CT 3'

3' TC|GA 5'
SmaI

5' CCC|GGG 3'

3' GGG|CCC 5'
HbaI

5' GCG|C 3'

3' C|GCG 5'

(The | symbol indicates the specific cut sites.)

Each answer may be used once, more than once, or not at all.

1.

5' -TAGACTGAATTCAAGTCA- 3'

3' -ATCTGACTTAAGTTCAGT- 5'
2.

5' -ATACGCCCGGGTTCTAAA- 3'

3' -TATGCGGGCCCAAGATTT- 5'
3.

5' -CAGGATCGAAGCTTATGC- 3'

3' -GTCCTAGCTTCGAATACG- 5'
4.

5' -AATAGAATTCCGATCCGA- 3'

3' -TTATCTTAAGGCTAGGCT- 5'
5.

Which restriction enzyme(s) is(are) 4-cutters (i.e. they recognize and cut at 4-bp sequences, instead of 6-bp sequences)?
6.

Which restriction enzyme(s) do not leave a "sticky end" after cutting the DNA?
A.

Alu
B.

AluI, SmaI
C.

All 6 of these restriction enzymes.
D.

None of the 6 restriction enzymes listed.
E.

BamH1
F.

HindIII, AluI
G.

EcoR1
H.

SmaI
I.

SmaI and HbaI
J.

HbaI
K.

HindIII
L.

AluI, HbaI
M.

EcoRI, AluI
N.

AluI
O.

BamHI, AluI

A-Which of the following is NOT one of the steps required toclone a piece of DNA using a plasmid vector?
1. Cut a vector and a DNA insert with aspecific restriction enzyme to create complementary "stickyends."
2. Isolate DNA fragments by gelelectrophoresis.
3. Ligate insert DNA and a vector withsimilar sticky ends together.
4. Transform ligated pieces of DNA into E.coli.
5. Select the plasmid vector that hasreplaced its origin of replication with insert DNA.

B-A representative genomic library of anorganism's DNA fragments results from the use of
1. DNA ligase joining multiple types ofvectors.
2. partial digestion with frequentlycutting restriction endonucleases.
3. many pieces of recombinant DNA fromseveral different types of organisms.
4. viruses that cleave the DNA into randompieces.

C-One of the most usefulmethods for identifying a specific DNA sequence is
1. the Southern blot.
2. the Western blot.
3. magnetic resonance imaging.
4. the Eastern blot.
5. thin-layerchromatography.