|Now you've had some practice using the database, and a web-based restriction
enzyme analysis program. Let's talk about some of the properties of restriction enzymes,
and in particular the types of ends they leave after cleavage.
|Recognition sequence and DNA ends
Take a look at the following examples of DNA restriction enzyme
Kas I (G^GCGCC)
Nar I (GG^CGCC)
Ehe I (GGC^GCC)
Bbe I (GGCGC^C)
You see that the same sequence is recognized by four isoschizomers
that break the phosphodiester backbone differently. The ends generated by these four
would consequently be different:
Kas I NNNNNG GCGCCNNNNNN
Nar I NNNNNGG CGCCNNNNNN
Ehe I NNNNNGGC GCCNNNNNN
Bbe I NNNNNGGCGC CNNNNNN
The point here is that enzymes leave different types of DNA ends, and this is a matter
that is independent of recognition sequence. In the example above, a digestion product
using Kas I would not be compatible with a digestion product of Nar I, because they
could not hydrogen bond.
Kas I NNNNNG CGCCNNNNNN Nar I
Bbe I has a GCGC-3' overhanging end, and similarly it cannot anneal
to any of the other three examples. It does have ends that are compatible with ends
generated by Hae II (RGCGC^Y) however. Here is an example:
Hae II NNNNNAGCGC CNNNNNN Nar I
In this situation, the ends would match perfectly and the phosphodiester
bonds could be sealed with the enzyme T4 DNA ligase.
Would NNNNNAGCGCCNNNNN be a Hae II site anymore?
Would the ligated sequence NNNNNAGCGCCNNNNN be a Nar I site anymore?
Are there any HaeII-NarI site fusions that would preserve both enzyme
sites in the ligated product?
|Blunt ends: the great equalizer
Blunt ends are always compatible with each other, because there
are no H-bonds being formed that would define compatibility or incompatibility. So,
a DNA end generated by Ehe I is compatible with a DNA end generated by EcoRV (GAT^ATC):
Ehe I NNNNNGGC ATCNNNNNN EcoRV
This is a mixed blessing, for while the ends will always fit together there is a
lack of specificity in assembly. Having cohesive ends gives better control of the
assembly process because you can force the DNA fragment to be inserted in a single
orientation. For example:
BamHI BamHI EcoRI EcoRI
NNNNNNG GATCCNNNNNNNG AATTCNNNNNNNNN
NNNNNNCCTAG GNNNNNNNCTTAA GNNNNNNNNN
In this example, the green DNA fragment (center) can only be inserted
with the BamHI site on the left and EcoRI site on the right. This is called forced cloning, and it is not possible
when the ends are blunt.
We can make a cohesive end into a blunt end using DNA polymerases
such as Klenow (fragment of E. coli DNA polymerase I), T4 DNA polymerase, or Pfu
polymerase. Let's review:
In this case, a 5' overhanging end is being filled in with newly-synthesized DNA.
Restriction enzymes typically leave small overhanging ends, and they are usually
of the 5' overhanging type.
Bam HI NNNNNNG GATCCNNNNN
Fill in one G nucleotide, and you have:
Fill in the next A nucleotide, and you have:
Then the next T nucleotide, and you have:
Finally the next C nucleotide, and you have a blunt end:
Now the enzyme cannot add additional nucleotides to the 3' end
because it requires a template:
If there is a 3' overhanging end, then the 3' to 5' exonuclease removes it, leaving
a blunt end also.
Here are some examples of what the enzymes mentioned earlier (Klenow, T4 DNA polymerase,
or Pfu) would do to ends left by the four restriction enzymes mentioned earlier:
Kas I NNNNNG GCGCCNNNNNN
...would be filled in to yield:
Nar I NNNNNGG CGCCNNNNNN
...would be filled in to yield:
Ehe I NNNNNGGC GCCNNNNNN
...would be unchanged
Bbe I NNNNNGGCGC CNNNNNN
...would be subject to the 3'-5' exo, leaving:
Having modified the DNA ends left by these four enzymes, all are now mutually compatible,
and would be compatible with other blunt ends. Note that where modifications have
taken place, the enzyme site is generally destroyed upon religation. Sometimes that's
exactly what you want.
Here is a protocol for Klenow treatment, downloaded from the Fermentas Inc. site
Protocol for Filling-in Recessed 3'-termini of Double-stranded
DNA (with Klenow Fragment)
1.Dissolve 0.1-4µg of digested DNA in
10-15µl of water.
10X reaction buffer 2µl,
2mM 4dNTP mix 0.5µl (0.05mM - final concentration),
Klenow fragment 1-5u,
deionized water up to 20µl.
3.Incubate the mixture at 37°C for 10 minutes.
4.Stop the reaction by heating at 70°C for 10 minutes.
1.Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John
Wiley & Sons, Inc., Brooklyn, New York, 3.5.7-3.5.10, 1994-1997.
You may also be interested in reading through the
kit instructions for the Fermentas DNA Blunting and Ligation kit.
This site explains some of the content that has been discussed to this point.
|Putting it together - the right way.
As we've discussed in class, we use the enzyme
T4 DNA ligase to make covalent connections in the phosphodiester backbone. It was
indicated in a previous lecture that 5' ends of DNA usually have a phosphate group,
and we know that the phosphate group is required for ligase activity (as is ATP as
a source of energy). We've also already discussed an enzyme (T4 polynucleotide kinase)
that can be used to add a 5' phosphate where one is lacking, for example on a PCR
oligonucleotide primer. When DNA is treated with the enzyme alkaline phosphatase,
the 5' phosphate groups are removed.
Activity of alkaline phosphatase - removal of 5' phosphates
Here's a nice application: If a linearized vector
is dephosphorylated in this way, it cannot reclose upon itself because the enzyme
T4 DNA ligase requires that a 5' phosphate group be present. A DNA fragment that
has 5' phosphates still present can form a bridge between the dephosphorylated ends,
so insertions are favored! When you are trying to combine two molecules, this removal
of 5' phosphates from the vector (alone) keeps it from reclosing on itself and spoiling
Preventing reclosures by use of a dephosphorylated vector
What you get in the end: There are two widely separated nicks in the final product, because
two of the four ligation events were prevented by the lack of 5' phosphates. Still,
two out of four is good enough! The bacteria will fix the remaining nicks after the
DNA is transformed.
Two sources of alkaline phosphatase are commonly
used for this work:
The shrimp alkaline phosphatase is heat sensitive
(it is derived from an Arctic shrimp that loves the cold!), so the enzyme can easily
be inactivated at a moderately high temperature (65 degrees, 15 minutes). The calf
intestinal alkaline phosphatase is relatively stable, so it must be inactivated at
higher temperature, or via digestion with proteinase-K enzyme.
Why is it so important to inactivate the alkaline phosphatase enzyme? Because if
it contaminates your ligation reaction, it will strip the 5' phosphates off of the
DNA insert as well. That will block all ligation events, including the ones you want!
Here is a protocol for CIAP treatment, downloaded from the Fermentas Inc. site
Protocol for Dephosphorylation of DNA 5'-termini
(with Calf Intestine Alkaline Phosphatase)
1.Dissolve DNA (1-20 picomoles of DNA termini) in 10-40µl deionized water.
2.Prepare reaction mixture by adding the following:
DNA solution 10-40µl,
10X reaction buffer 5µl,
deionized water to 49µl,
alkaline phosphatase 1u/µl.
3.Incubate at 37°C for 30 minutes.
4.Stop reaction by heating at 85°C for 15 minutes or extract DNA with phenol/chloroform
and then precipitate with ethanol.
Dephosphorylation can be performed by adding calf intestine alkaline phosphatase
directly in mixture after DNA cleavage with a restriction endonuclease. We recommend
using 0.05 units calf intestine alkaline phosphatase for dephosphorylation of 1 picomole
DNA termini. The enzyme can be diluted with 1X reaction buffer.
Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M. et al., ed.), John
Wiley & Sons, Inc., Brooklyn, NY, 3.10.1-3.10.2, 1994-1997.
|The reclosure problem and the statistics of ligation
The method just described, of treating a vector with CIAP, is one
of many that are used to prevent reclosure of a plasmid vector without inserted DNA.
The process of combining two pieces of DNA (a bimolecular ligation) is tricky because
it is often the case that the two ends of one piece of DNA are closer to each other
in solution than DNA ends from two different molecules. Why? Because the two ends
of a single DNA molecule are tethered to each other through the DNA molecule. On
the other hand, in a dilute ligation reaction the two ends of different DNA molecules
may rarely bump into each other.
If you attempt to reclose a plasmid by ligation,
what are you actually doing? You are asking that the two ends of the DNA "find
each other" in the solution, and that T4 DNA ligase covalently connect the phosphodiester
backbones (using a bit of energy taken from an ATP molecule).
The question you may then ask, is:
"How easy is it for two DNA
ends to find each other in solution?"
To answer that, we need to delve into the subjects
of public drunkenness and physical chemistry. Come to think of it, those are pretty
much the same thing. Picture in your mind a drunken person, hanging onto a lamppost
for support. As he staggers away from the post, the direction of each step is random.
How far away from the lamppost will he tend to be if he has taken N steps? The path
of the person may look something like this, after 1000 steps.
Where R is the distance from the lamppost to the
drunk, we have the greatest likelihood that:
Where N is the total number of steps taken, and
is the square
root of the average squared step size or root mean squared step size. Of course the
result has a statistical outcome, and this is just the distance with the highest
probability in a distribution. If you move the drunk back to the lamppost and have
him try again, you will most likely get a different result each time.
A linear piece of DNA tends to spread out in solution
by a "random walk" of short segments in much the same way as the drunk
staggers away from the lamppost. The distance from one end of the DNA to the other
is most likely to be where
represents a characteristic (root mean square) step length
(called the "persistence length") which is related to the flexibility of
the DNA (its sequence characteristics) and the hydration of the nucleic acid (the
solution characteristics). If the DNA is more flexible, the step length is smaller.
the number of these segments or "steps" in the entire DNA.
In the case of our attempting to ligate DNA ends
in a single linear molecule, we want the two ends of a DNA molecule to "find
each other" in solution. That is analogous to expecting the drunk to find their
way back to the lamppost after taking N steps in random directions. The probability
of this happening is proportional to time (because the DNA is always changing conformation
and trying different "random walks") and the square root of the length
of the DNA (by the above equations).
Let us now attempt to sober-up!
So far we have been discussing
the behavior of a single linear DNA molecule, and I think you will agree that the
chances that one end of a single molecule will find the other end of the same
molecule do not depend on the concentration of DNA in the solution. On the other
hand, if we want the ends of two different DNA molecules to find each other
in solution, their independent concentrations will be extremely important! If the
concentration of either DNA end in solution is too low, the other end cannot find
it in a reasonable period of time.
Suppose you now want to insert a DNA fragment into
a linearized vector. This isn't quite the same situation as we had considered before,
because now the efficiency of the reaction will depend on DNA concentration. We want
to have inter-molecular ligation; ligation between the two different molecules. At
the same time, we want to avoid reclosure of the plasmid without an inserted DNA
fragment (intra-molecular ligation)
What are the chances of intra- vs. inter-molecular ligation?
If the DNA is very concentrated, inter-molecular
ligation is more likely. If the DNA is dilute in solution, then intra-molecular ligations
are more likely.
Therefore, the following
is sound advice:
- If you desire to circularize a single linear molecule,
the ligation should be performed at low DNA concentration.
- If you desire to combine two different DNA molecules
by ligation, the concentration of each should be high - on the order of 1 micromolar.
The optimal molar ratio of "insert" to "vector" is generally
taken to be about 2:1.
Remembrance of things past...
Do you remember in your old chemistry class, how
a careful distinction was made between the concept of "concentration" and
"activity?" You may recall that square brackets [DNA] were used
to indicate concentration in an equation, whereas round brackets (DNA) were
used to indicate activity. Here's where that little pearl of wisdom will finally
become useful! It is possible for us to change the solution characteristics so that
(DNA) > [DNA]. If we add polyethylene glycol (PEG) to a ligation reaction,
it ends up taking over and monopolizing some of the aqueous solution volume. The
DNA has less space, because it has to share the solution with the PEG molecules which
are real hydrogen bond hogs! Since the DNA has less space, it can find other molecules
of DNA more easily. Its concentration (i.e. moles per liter or grams per liter) hasn't
changed, but its "activity" is higher.
|A different trick
How do we solve the problem of reclosure, if we
don't want to treat the vector with alkaline phosphatase? Here's another trick that
works in some circumstances. If an unwanted ligation product (for example, a reclosure)
has a restriction enzyme site, and that site is not present in the desired product,
the unwanted product can be specifically linearized with the enzyme after
ligation is completed.
For example, suppose we were attempting to clone
a fragment that has Bgl II ends (Bgl II cuts at A^GATCT), into a vector that had
been linearized with BamHI (G^GATCC). These ends are compatible but their ligation
eliminates both the BglII and BamHI recognition sites.
The unwanted reclosure has a BamHI site!
The reclosed plasmid has a BamHI site (because
the two halves just reformed into a complete site), but the desired product does
not! If both are present in the ligated material, the reclosures can be specifically
linearized by treatment with BamHI. That is, their ligation can be effectively reversed!
|Yet another trick:
Here's a trick that works in some circumstances.
Suppose you've digested a vector with the enzyme XhoI (C^TCGAG), and you partially
fill in the overhanging 5' ends with the enzyme Klenow and the substrates dCTP and
dTTP. Note that the other two nucleoside triphosphates are excluded from the reaction.
Here is what would happen:
The Xho I "partial fill-in" reaction
|Before digestion with Xho I
|After digestion with Xho I
|After partial fill-in with dCTP and dTTP
Now you've created a two base 5' overhang that
is incompatible with itself, so it cannot reclose naturally. On the other hand, the
overhang is compatible with 5'-GA overhangs:
5' GA overhang
|BamHI end, partially filled in with dGTP and dATP
|Bgl II end, partially filled in with dGTP and dATP
|Sau3AI end, partially filled in with dGTP and dATP
|Bcl I end, partially filled in with dGTP and dATP
|Xho II end, partially filled in with dGTP and dATP
And so, if you prepare a DNA insert with one of
these enzymes and partially fill in the ends (as shown), the problem of reclosures
should be eliminated. Only the vector and insert ends can be ligated.
|Linkers and adapters can create a new restriction site
Suppose you have a collection of DNA fragments
with blunt ends, and you want to introduce them efficiently into a vector that is
linearized at an EcoRI site. From what you have learned so far, you might think that
the best thing to do would be to make the vector ends blunt as well so that they
will be compatible. You could...but that would not give you a high efficiency of
ligation (meaning fewer candidate colonies per transformation). It may also be the
case that you would like your product to retain EcoRI sites. If you make the vector
ends blunt, you will destroy those EcoRI sites.
One solution is to ligate a small double-stranded
DNA containing an EcoRI site to the end of the blunt DNA fragments. Since this small
DNA can be added to the ligation reaction in great excess, it is an efficient reaction.
Then the DNA can be digested with EcoRI to create the proper overhanging ends that
will be compatible with the vector. We call this small piece of DNA a linker.
Ligation and digestion of linker
Blunt end - before addition of linker
After ligation of linker
After digestion of linker
Note that if the linker has 5' phosphates (i.e.
if it is phosphorylated) then a great many linkers may be ligated to the fragment
in a tandem repeat. These will all be digested away by the enzyme, leaving only the
proximal sequence as shown.
One may also use a non-phosphorylated linker, in
which case only one ligation event will occur - between the 5' end of the fragment
and one of the 3' ends of the linker. In the example given:
After digestion of linker
If the linker had not been phosphorylated, the
GGCC of the lower strand would not be ligated to the NNNNNN because
would have lacked a phosphate at the time of ligation.
A problem exists with linkers however. What happens
if you add an EcoRI linker (as shown above) but your fragment has an EcoRI site right
in the middle of it? If you were to treat your ligated linker plus fragment with
the enzyme, you would digest your fragment right in the middle, which would probably
make you sad!
Adapters solve the problem, without need for digestion
An adapter is (at least in some terminologies)
a pair of non-phosphorylated single strand DNAs that hydrogen bond to create one
overhanging and one blunt end.
Ligation of non-phosphorylated adapter
Adapter andblunt end
The cohesive end is added automatically - there's
no need to treat the fragment with restriction enzyme, so internal sites are safe! These linkers and
adapters are some of the tricks of the trade for engineering DNA ends, but don't
forget that one of the most powerful ways of controlling DNA ends is a method we've
already discussed: The polymerase chain reaction.
Don't touch that dial! In the next lecture, we'll look at a few more
ways of controlling the ends of DNA, and controlling the ligation reaction without
using restriction enzymes or DNA ligase!