Download 10 Restriction Analysis of Genomic DNA

10 Restriction Analysis of Genomic DNA
Objectives:
A) To determine the rough location of restriction sites of an unknown
restriction enzyme and B) to use this information to determine the identity
of this restriction enzyme.
Introduction: Genomic DNA is very large. For example, the human genome contains
over 1 billion (109) base pairs. This is far too big to be analyzed at one
time in its entirety. Deoxyribonucleic acids can, however, be analyzed in
a variety of ways. The general strategy is to break up the DNA into
fragments of manageable size. One very useful means by which this is
done is to digest the DNA with a variety of enzymes known as restriction
endonucleases or restriction enzymes (REs).
REs are bacterial enzymes that cleave double-stranded DNA. There
are two types of restriction enzymes. Type I REs are important in
bacterial function but do not cleave DNA at specific sequences. Type II
REs require highly specific sites for DNA cleavage and are thus extremely
useful tools in molecular biology. These enzymes allow the cloning and
purification of defined DNA fragments. Restriction enzymes are typically
isolated from a variety of bacterial strains. For example, the RE Hind III
was originally isolated from Haemophilus (H) influenzaa (in) and Eco RI
was isolated from Escherichia (E) coli (co), an enteric bacterium.
Bacteriophage lambda (λ) is essentially a virus that infects E. coli
bacteria. The genome of bacteriophage λ is a double-stranded DNA
molecule approximately 50kb in length. Bacteriophage λ has been
extensively characterized, including exhaustive restriction analysis.
Consequently, the number of times each RE cuts bacteriophage λ's
genome is known and is easily found in any biotechnology company's
catalog. The genomic DNA to be used in today's experiment was isolated
from this virus.
In today’s experiment, λDNA will be digested with a restriction
enzyme. The restriction pattern then will be electrophoretically analyzed,
compared to standards, and the identity of the unknown restriction enzyme
determined. As an aid in determining the length of time the gel should be
run, a tracking dye will be added to the samples. This dye is very similar
to that used in Chapter 7. The mixture contains bromophenol blue (BPB;
dark blue color) and xylene cyanol (XC; medium sky blue). The BPB
runs at a rate that is approximately equal to a 500-bp DNA fragment; XC’s
migration is similar to a 4000-bp fragment.
Procedures:
The procedures for this experiment vary slightly from semester to semester.
Your instructor will give you the exact protocol in the pre lab lecture.
The general procedure will be to add the appropriate buffer to the DNA sample.
Different enzymes require different buffer conditions, pH, salt concentration, and the type
of buffer (phosphate verses acetate for example) all are important to enzyme activity.
The wrong buffer may cause the enzyme to be totally inactive or it may cause the enzyme
to recognize and cut at unanticipated sites in the DNA. This erroneous activity is called
star activity.
Addition of the enzyme is important as well. These enzymes are stored in glycerol so
that they can be kept at -20°C without freezing solid. Excess glycerol can also cause
inactivity or star activity. Typically keep enzyme volume less than 10% of total reaction
volume. Typically we add only 1 µL of enzyme. Thus a drop of enzyme on the outside
of the pipette tip can dramatically alter the amount of enzyme (and glycerol) that is
added.
After addition of enzyme and buffer, the samples are heated to 37°C for some length of
time. Most restriction enzymes work best at 37°C but some require other temperatures
(Apo I requires 50°C and Bae I requires 25°C). Information about the temperature and
buffer conditions will be provided by the supplier of the enzyme.
During this incubation time you can prepare the agarose gel.
Prepare a 20-mL, 0.7% agarose gel containing 0.5µg/mL EtBr in 1 X TAE
buffer. The calculations for making this gel are very similar to those used in
Chapter 7. The EtBr stock is 10mg/mL.)
After the incubation loading dye will be added to the mixture. The loading dye serves
two functions, the dye allows us to judge how far the DNA has migrated in the gel. The
dye is also negatively charged so it moves in the gel. We use Bromophenol blue which
migrates as if it were about 400 bp.
The gel is then visualized with UV light.
By comparison of the known molecular weight standards with the fragments in your
restriction digest you can estimate their sizes.
We use the 1 kB ladder from New England Biolabs. The sizes are as follows:
10 Kilobases
8
6
5
4
3
2
1.5
1.0
0.5
Use this information to help you determine which enzyme you had. It was one of the five
listed on this page.
These are NOT the sizes you would expect to see!
These are positions or Sites where restriction enzymes cut phage lambda phage:
Bgl II: 415, 22425, 35711, 38103, 38754, and 38814
EcoRI: 21226, 26104, 31747, 39168, and 44972
HindIII: 23130, 25157, 27479, 36895, 37459, 37584, and 44141
MluI: 458, 5548, 15372, 17791, 19996, 20952, and 22220
NdeI: 27630, 29883, 33679, 36112, 36668, 38357, and 40131
Lamda phage DNA is linear and 48502 bp long
Consider if you have a yard stick and you break it at the 10, 18, and 30 inch marks (Sites
or Positions) what size pieces will you have?
Electrophoretic Pattern of λ DNA
Standards Und. B I B II
E
H
M
N
P
Und: undigested DNA; B I: Bgl I; B II: Bgl II; E:
Eco RI; H: Hind III; M: Mlu I; N: Nde I; P: Pst I
Make a sketch of the gel for your notebook and hand in the actual photo with your
lab report:
Restriction Analysis of Genomic DNA STUDY GUIDE
1.
What is bacteriophage λ?
2.
What are restriction enzymes?
3.
What are the nucleic acid structural features recognized by the restriction enzymes
used in this experiment?
4.
Towards which electrode (cathode or anode) do nucleic acids migrate? Why?
5.
The tracking dye contains two different dyes.
a.
What are the names of the two dyes?
b.
Which of these dyes will migrate the fastest in the electrophoretic field?
6.
Why was an undigested DNA sample run on the gel?
7.
_______ is used as an aid in visualizing the DNA with ultraviolet light.
a.
Tracking dye
b.
STET
c.
Ethidium bromide