Biology 2581B Study Guide - Midterm Guide: Sticky And Blunt Ends, Recognition Sequence, Restriction Digest

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6 Mar 2017

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Restriction enzymes: restriction enzymes recognize and cut specific sequences of dna, on(cid:272)e (cid:272)ut, the dna (cid:272)an produ(cid:272)e 3" sti(cid:272)ky ends, 5" sti(cid:272)ky ends, or (cid:271)lunt ends (cid:449)ith no o(cid:448)erhang. The sequence detected, cut performed, and ends produced are all specific to the restriction enzyme used. Different enzymes can recognize different sequences and produce identical overhangs. Different enzymes can recognize the same sequence and produce different overhangs: digested fragments can only re-anneal if they have the same type of overhang with complementary sequences. Blunt ends can be ligated to any other blunt end. But you will end up losing the recognition site: applies to the sticky ends as well, depending on the length of the recognition site, dna can be digested to yield different results. Gel electrophoresis agarose gel separate dna fragments by size: dna is negatively charged, with a uniform mass-to-charge ratio.

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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 Helenâs conceived child: Child ____

b. George and Helenâs conceived child: Child ____

c. George and Helenâs adopted child: Child ____

rosecrab387

1) Which of the following is FALSE about plasmid vectors ued in recombinant DNA research?
A) Usually carrry an antibiotic resistance gene
B) Clonin sites are always located between genes, that is, outside coding sequences
C) May hae a multiple clonin gsite which can be cut by several restriction enzymes
D) May be expression vectors

2) Which of the following is FLASE about Sanger Dideoxy DNA sequencing protocols?
A) Different specfiic chemical reactions break the DNA at each base.
B) Fluorescent molecules can be used to detect the DNA
C) The Template is single stranded DNA
D) Many steps can be automated

3) A southern blot transfers ___ to a Nitrocellulose membrane.
A) Protein
B) RNA
C) DNA
D) Lipid

4) Which of the following is true about cDNA?
A) It is made from DNA template
B) It is isolated from bacteria
C) Reverse transcriptase is used to synthesize cDNA
D) It has introns

5) The restriction endonucleases used in recombinant DNA work:
A) Always produce blunt ends (No unpaired bases)
B) Recognize sequences 4-6 bp long
C) Cut the DNA outisde the recognition sequence
D) All of the above

S17

THANK YOU VERY MUCH!!!

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

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Biology 2581B Study Guide - Midterm Guide: Sticky And Blunt Ends, Recognition Sequence, Restriction Digest

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