Biochemistry 2288A Lecture : Restriction Enzymes

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orchidotter238

2 Jan 2012

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Key aspect of letting molecular bio startwithout it, we wouldnt have recombinant techniques. Restriction enzymes can be thought of as site specific dna scissors. They recognize specific sequences in dna and cut it. Dna is a very long (megabase pairs), relatively homogenous polymer. It is made up of same four base pairsits big and homogenous. To manipulate it, you need ways to cut dna into defined fragments. Restriction enzymes will allow dna to be broken up into defined workable units. Its easy to break dna into random unitsbut we want specific cuts out of dnaso were working with defined unitsr enzymes will do this. Restriction enzymes allow dna to be broken up into defined workable units. Def: site specific dna binding protein that recognize and cleave palindromic dna sequences of 4, 6, 8 base pairs. They are dna binding proteins just like trp repressor, lac repressor, capall dna binding proteins.

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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

sangriapug213

Can sticky ends formed by one specific restriction enzyme become a restriction site of another restriction enzyme?

Restriction enzymes are used to construct restriction maps ofDNA. These are diagrams of specific DNA molecule that show thesites where the restriction enzymes cleave the DNA. To construct arestriction map, purified samples of the DNA are treated withrestriction enzymes, either alone or in combination, and then thereaction products are separated by agarose gel electrophoresis, atechnique that separates the DNA fragments based on size. (Thesmaller fragments travel more quickly and are found near the bottomof the gel, whereas the larger fragments are found near the top ofthe gel.)

Use the results of the agarose gel electrophoresis separation(above) to construct a restriction map for the sample of DNA.

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Biochemistry 2288A Lecture : Restriction Enzymes

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