Analise_de_restricao_Exercicio

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Restriction Analysis
Eva Corinna Feil, Eva-Maria Neher, XLAB, Göttingen
Restriction Enzymes 
Restriction enzymes are very important enzymes that are vital to our manipulation ofDNA. Restriction enzymes are proteins produced by bacteria to prevent or restrict
invasion by foreign DNA. They act as DNA scissors, cutting foreign DNA into pieces so
it cannot function. Restriction enzymes recognize and cut at specific sequences of
nucleotides in the DNA molecule, called restriction sites. Each different restriction
enzyme (and there are hundreds, made by many different bacteria) has its own
restriction site.
A typical restriction site is 4-6 base pairs (bp) long. It is a palindrome; that is, it reads
the same backwards and forwards. Along a DNA molecule, a specific combination of 4
bp occurs randomly once every few hundred bases. A specific sequence of 6 bp occurs
randomly once every few thousand bases. Some DNA
molecules have many sites for a particular restriction
enzyme; others have none. When restriction enzymes
cut DNA into pieces, the sizes of the DNA fragments
correspond to the distances (in base pairs) between
restriction sites. Restriction enzymes are named for the
bacteria that produce them. SspI comes from the
bacteria Sphaerotilus species (the Roman number I in
the enzyme’s name shows that this was the first
restriction enzyme found in this organism.) Pstl was the
first restriction enzyme isolated from Providencia stuartii. And Hpal was the first
restriction enzyme isolated from Haemophilus parainfluenzae.
Restriction Maps and Logic Puzzles
A new DNA molecule is like uncharted territory to a scientist. Like any explorer, the first
thing a scientist does with new territory is to map its important features, such as size
and the relative locations of certain parts. Scientists have a unique set of tools to map
new DNA. Maps of DNA are called restriction maps, named for the enzymes used as
mapping tools. Restriction enzymes recognize and cut DNA at specific sequences of
nucleotides (called restriction sites), and the sizes of the DNA fragments correspond to
the distances (in base pairs) between restriction sites. Every time the same piece of
DNA is cut with a given enzyme, the same fragments are produced.
Working out the locations of the different restriction sites is a problem in logic. Doing a
restriction enzyme digest with a single enzyme, for example, only tells you how many
sites are present for that enzyme. Doing restriction digests with more than one enzyme
at a time can give clues as to where those restriction sites are in relation to each other.
In the logic puzzle below, you have a series of step-by-step questions to help you make
a restriction map of unknown DNA. First, you examine a mock gel to compare the DNA
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fragments from restriction
digests of the unknown DNA
to DNA fragments of known
sizes. 
Figure 1 shows lambda
DNA digested with the
restriction enzyme Pstl to
produce DNA fragments of
known sizes. Use the sizes
of the known DNA
fragments to estimate the
sizes of the unknown DNA
fragments. Then, add up the
sizes of the individual
fragments from each
restriction digest to
determine how large the
original uncut piece of DNA
was. Next, determine the
number of times each restriction enzyme cut the DNA, and the distance between the cut
sites. Finally, determine where the cut sites are relative to one another.
Four different restriction enzyme digests have been done on our unknown DNA (Fig. 1).
Three restriction enzymes have been used-Hpal, PstI, and Sspl.
Samples of our DNA have been incubated with either Hpal alone; Hpal and PstI; Hpal
and Sspl; or Hpal, PstI, and Sspl. The single restriction enzyme digest helps you deduce
the number of cut sites present; the double and triple digests allow the cut sites to be
mapped relative to each other.
Questions
1. Estimate the sizes of the DNA fragments in base pairs (bp) by comparing them 
to the labeled lambda/Psd -size markers. These sizes do not have to be exact. 
Sizing of the smaller fragments is more accurate than sizing of the larger 
fragments.
2. Determine the total size of the digested DNA by adding up the sizes of the
fragments from each digest. Take an average size from the 4 digests. Remember,
the same DNA was digested in each sample, so the fragment sizes from the
different digests should always add to the same total.
3. There are 2 Hpal sites present. Based on the number of fragments obtained from
the Hpal digest, is this DNA linear or circular? Draw the DNA with the Hpal sites
present.
Figure 1
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4. How many PstI sites are present?
5. Where is the PstI site? Draw the position of the PstI site on the plasmid, relative to
the Hpal sites. Remember, a plasmid is just a small, circular piece of DNA.
6. How many SspI sites are present?
7. Where is the SspI site? Draw the position of the SspI site on the plasmid, relative
to the Hpal sites. It might be best if this is done in a separate sketch from the PstI
site sketch, because we have not yet determined where the SspI and PstI sites are
relative to each other.
8. Will the 600-bp Hpal fragment remain unchanged after digestion with either PstI or
Sspl? (Check Fig. 1).
9. Which fragments are unchanged from the Hpal/Pstl digest to the Hpal/Pstl/SspI
digest? Which fragments disappeared? Why did those fragments disappear?
10. Which fragments are unchanged from the Hpal/SspI digest to the Hpal/Pstl/SspI
digest? Which fragments disappeared? Why did those fragments disappear?
11. Is there a fragment that appears only in the Hpal/Pstl/SspI digest? What does this
mean?
12. Draw the full plasmid map, with all restriction enzyme recognition sites present in
their relative locations.
Answers
1. Hpal Hpal/PstI Hpal/SspI Hpal/Pstl/Sspl
300 300
600 600 600 600
1200 1200
1800
2100
3000
3300
2. 3,900 base pairs (3.9 Kb)
3. This is circular DNA (plasmid). Restriction mapping
can certainly be done on linear DNA; however, it is
slightly more complex and calls for a different set of
restriction enzyme digests.
4. Since there are 2 fragments after digestion with Hpal and 3 fragments after
digestion with Hpal and PstI, there is one Pstl site.
5. The Pstl site is within the 3,300 bp Hpal fragment.
6. Since there are 2 fragments after digestion with Hpal and 3 fragments after
digestion with Hpal and Sspl, there is 1 SspI site.
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7. The SspI site is in the 3,300-bp Hpal fragment.
8. Yes. No SspI or Pstl sites are present within this fragment.
9. 600 and 1,200 bp. The 2,100-bp fragment disappeared because it contains a SspI
site.
10. 300 and 600 bp. The 3,000-bp fragment disappeared so it must contain a Pstl site.
11. Yes. That there is a fragment with a SspI site on one end and a Pstl site on the
other end.
12. Full-plasmid map: You may draw either of the 2 options; they are simply mirror
images of one another.