Some commonly used restriction enzymes

 

[ ] BamH1, EcoRI, HindIII, KpnI, PstI and SphI. Their specific DNA recognition site is generally shown like this: for example BamHI, here, cuts between the G’s in the site GGATCC. Only one strand of the double stranded DNA site is shown, and the orientation is always like this: 5’ to 3’ is from left to right.

The double-stranded recognition sequence is shown in the middle column of this picture.These restriction enzymes recognise so-called palindromic sites, which means that at the recognition site, the complementary strands are identical: in this EcoRI site for example, it reads from 5' to 3' : GAATTC here and in the opposite strand from 5' to 3' the same sequence: GAATTC. So, for example for HindIII [ ], it reads: a-cut-a-g-c-t-t, it means that the enzyme cuts the double-stranded DNA like this [ ], and the resulting DNA ends look like this [ ]. These are 5' ends [ ], they overhang the 3' ends, here [ ]. As a result, the DNA ends generated by the enzymes shown here, are staggered: each DNA strand has overhanging, and recessed ends.

BamHI, EcoRI and HindIII have 5’ overhanging, or: 3’recessed ends. They differ with the other enzymes here, KpnI, PstI and SphI. Those enzymes give 3’ overhangs, and 5’recessed ends. For example: Kpn I: this [ ] is the recognition site g-g-t-a-c-cut-c, it cuts like this [ ], and the ends look like this, with here [ ] their 3'overhangs.

The overhangs created by a particular restriction enzyme are self-complementary, because of their palindromic nature. That's why they are called "sticky" or "cohesive". They are used very efficiently in ligation steps in cloning procedures.

Some other restriction enzymes give so-called blunt ends, with no overhangs. These enzymes are not shown in this movie.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This movie is about Partial digestion.

Restriction enzyme digestions are usually done under optimal conditions to fully digest the DNA, but is some cases you need a DNA fragment that can only be obtained by partial digestion of the DNA.

This movie is about such an experiment. You will see how we cut plasmid DNA to get a vector fragment usefull for another subcloning experiment.

On the left you see a map of the intact plasmid vector. By digestion of Acc I and Pvu II we got the large vector fragment we needed. It’s highlighted on the right.

Problem was that this fragment still contained a Pvu II site, so to obtain the vector fragment we needed we should leave this site intact.

We will now show you how we set up the digestion reaction.

This is the pipetting scheme. We added 10 units of Acc I to cut 2 micrograms of DNA. This was enough restriction enzyme for a complete digestion. After one hour of Acc I digestion we added a small amount, one unit, of Pvu II. So we expected the Pvu II digestion to be only partial. This means that not all molecules would be cut at both Pvu II recognition sites. We would get some molecules only cut at this Pvu II site, and some others cut at only that Pvu II site. The exact result of a partial digestion reaction is hard to predict so we took samples at 5, 10, 17, 25 and 35 minutes after adding Pvu II. 4 μl samples were added to 16 μl of a 10mM EDTA solution. This would stop the restriction enzymes in the digestion reaction immediately. Then we ran the samples 1 to 5 in an agarose gel to separate the various DNA fragments. Before we will show you the gel result, we will first show you the fragments we expected to see.

 

Here you see the sizes of fragments we expected after complete digestion with Acc I and partial digestion with Pvu II. All DNA molecules were cut by Acc I and we could find molecules with additional Pvu II cuts either at the 69 site or at the 415 site, or of course at both sites. This would result in the six fragments shown here.

 

·         This fragment is the resukt of cutting by Acc I only.

·         These fragments are from an additional cut at the Pvu II 69 site laeving the Pvu II site at 415 intact.

·         These fragments are from an additional cut at the Pvu II site at 415 leaving the 69 Pvu II site intact.

·         Cuts at all three sites would give these fragments.

 

The 2626 basepair fragment was the one we needed. It’s the large fragment spanning from the 415 Pvu II site to the 180 Acc I site.

 

Now look at the gel result.

Here you see the black-and-white photograph of the gel after we stopped the electrophoresis. Here in lane 1 we ran a Lambda DNA Hind III digest as a size marker. The sizes are indicated at the left. The 4 large vector fragments migrated almost to te very end of the gel between these marker bands. This was close to the maximal separation we could get. The two smaller fragments have run out of the gel. Band 3 is the fragment we needed. DNA molecules cut only at the Acc I site are in this band. The lower band here comes from cutting at the AccI site and both Pvu II sites. As you can see longer incubation with Pvu II gives more of the lower band. The bands in between come from one Pvu II cut only. The 5 and 10 minutes digestions in lanes 2 and 3 give the largest amounts of these fragments. Band 3 was cut ot from those lanes and we purified the vector fragment from the gel slice. We used it in a ligation step for a subcloning experiment and in the end we got the clones we needed.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Making the sticky ends of DNA blunt
by using Klenow DNA polymerase

Restriction enzymes can generate three types of DNA ends:

either blunt ends

or two types of sticky ends:

with a 5' overhang, or with a 3' overhang.

Alternatively, one might say: with a 3' recessed end or with a 5' recessed end.

The overhanging single-stranded parts, here [ ] and here [ ] are generally 2 to 4 nucleotides long.

 

Imagine that for a particular DNA ligation, you need to modify the sticky ends of a DNA fragment into blunt ends. In that case, you could use the Klenow DNA polymerase enzyme to fill-in recessed 3' ends to form a blunt end,

the highlighted 4 nucleotides here [ ] are newly incorporated by Klenow polymerase.

Also you could use Klenow to remove the 3'overhang [ ]of the other type of sticky ends to get a blunt end.[ ]

 

First you should know a few things about the DNA polymerase used:

The polymerase is from E.coli. and it has three activities:

a 5->3' polymerase act

a 5->3'exonuclease activity and a 3->5' exonuclease activity.

 

The Klenow enzyme is the same enzyme, but without the 5->3'exonuclease activity.

It still has the 3->5' exonuclease activity and the 5->3' polymerase activity on primed DNA templates.

 

So, the polymerase activity will synthesize DNA . The direction is always from 5 to 3' [ ]

And with it's exonuclease activity the enzyme can chop off nucleotides from 3' DNA ends, but it leaves 5' DNA ends intact.

 

Let's look at this EcoRI example: the two ends shown here, can form the primed template for a so-called filling-in reaction with Klenow polymerase: here[ ] and here[ ] four nucleotides can be built in by the enzyme.

This is the result of the filling-in : with four nucleotides added to the 3’recessed ends, the double-stranded ends are now blunt.

You may apply the filling-in reaction mixture to an agarose gel, or purify the DNA by ethanol precipitation or by a quick and modern spin column-based procedure.

 

Let's go to a second example, KpnI: this enzyme gives a 3'overhang:

Blunting by filling-in is not possible, since DNA synthesis by polymerases is always from 5' to 3'. So, the 3' overhang has to be removed.

This can be done by the 3' to 5' exonuclease activity of Klenow polymerase, despite the fact that the exonuclease continues  in the double-stranded portion of the DNA molecule.

The reasonis  that the 3 to 5' exonuclease activity is much lower than the 5 to 3' polymerase activity, which is in fact the trick in this procedure: The exonuclease chops off not only the overhang, but it continues. But since the resulting 3' recessed ends are immediately filled in by the higher polymerase activity, the result is a perfect blunt end as you can see here below.

 

So, [ ], in the case of Kpn I, this is what you get: these [ ] sticky ends are converted in the blunt ends shown here [ ] by chopping off these overhanging parts.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In vitro Ligation

This movie is about ligation. In vitro ligation of DNA fragments generated by restriction enzymes. We'll show you what happens when fragments having cohesive ends are joined by ligation.

Here you see a part of two different DNA fragments, shown in green and red. The two strands of the double-stranded DNA are complementary and in the opposite orientation. They are joined by the hydrogen bonds [   ] between the bases..
The  green and red DNA fragments are the result of cutting with a particular restriction enzyme. Only one end of each fragment is shown, here in the middle [   ]. Restriction enzymes give DNA ends with a phosphate group at the 5' end  [  ] and a free hydroxyl group at the 3' end [   ]. Those groups can be covalently joined by the enzym DNA ligase, giving a so-called phosphodiester bond. Such a bond covalently joines two DNA ends. It can be made by the ligase protein molecule only when the phosphate and hydroxyl groups are directly adjacent. 

The sticky ends of the green and red fragments were created by the same restriction enzyme, so the overhanging single stranded parts, here and here [  ] are complementary. They can form a duplex, shown here [   ]. This duplex is not very stable because it is formed by a few hydrogen bonds only, shown in yellow here [   ]. The duplex holds long enough for the ligase to catalyze the ligation reaction. By the way, also blunt ends can be joined by ligase. But because no duplex can be formed the ligation rate of blunt ends is very low.
In this part [   ] of the picture the DNA is shown after the action of the ligase. The DNA ends are joined covalently and a the green and red fragment are forming one DNA molecule now..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ligation to construct a recombinant plasmid

In the laboratory situation, mostly one of the fragments to be ligated is the plasmid vector fragment, drawn here in green, having a replication origen [ ], and the other one is the insert (or 'donor') fragment, in red [ ].

Often there are several different donor fragments in the ligase reaction mixture. You should realise here that linear molecules will never give transformants, due to breakdown by exonucleases in the bacterial cell. Joining a vector and a donor fragment gives the desired recombinant DNA molecules, one is drawn here [A]. The formation of such a molecule in the ligase reaction mixture is a matter of chance. All DNA ends generated by the same restriction enzyme can be joined by the ligase enzyme. So, many undesired combinations will be formed. Most of them will not give transformants when you select those on the presence of a plasmid, for example by growing the bacteria after transformation on a medium where they need expression of a plasmid gene.

Circularised combinations of donor fragments only [B], will be lost, since they do not contain the replication origen and will not replicate.
Combinations which include more than one vector molecule will not efficiently replicate, when the replication origens are in the opposite direction, as drawn here, which is an inverted repeat. Transformants harbouring dimer plasmids, as direct repeats, can sometimes be found. They are not shown here 

When you want to get the desired recombinant by analyzing a number of randomly picked transformants, the most optimal ligation conditions should include:

[ ] no possibility for the vector molecule alone to ligate to a circulair form, so: no 'self-ligation' should be possible

[ ] a vector/donor molar ratio of about 1 to 3

[ ] a DNA concentration of 10 to 50 µg/ml

If you would not comply with these tips you may end up having to screen very many transformants before you find a single recombinant

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is a movie about how to purify a DNA fragment
from agarose gel.

Cut out the desired fragment band  from the gel after separation by electrophoresis. This is done on a UV lamp.

Transfer the gel slice to a fresh tube. Add 2.5 to 3 volumes of 6M Sodium Iodide and incubate the tube at 50°C untill the gel is completely dissolved.

Add a small amount of a suspension of very fine glass particles to adsorbe the DNA, which is now free in solution.

After centrifugation, wash the glass particles pellet, which now also contains the DNA, with cold 50% ethanol. This will remove any remaining iodide and keeps at the same time the DNA bound to the particles.

Remove the ethanol supernatant after pelleting the particles again. Resuspend the pellet  in a small volume of water or low salt buffer. This is to release the DNA from the glass particles.

Do a final centrifugation step, and transfer the supernatant containing the DNA molecules to a fresh tube

Your purified DNA fragment is now ready for further processing.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This movie is about Selection of Recombinants.


Recombinants are bacterial clones harbouring a recombinant plasmid. The original plasmid pBR322 is shown on the left. It has two antibiotic resistance genes. One for ampicillin [here] and one for tetracyclin [here]. You will now see an example where plasmids are selected in which a recombination has occurred. In this case it’s an insertion of a stretch of DNA in the tetracyclin resistance gene shown here. The insertion or recombination disrupts the tetracyclin resistance gene. The ampicillin resistance gene is kept intact in this experiment. The selection takes place after the transformation of the bacterial cells with the plasmid which was present in an in vitro ligation mixture. The bacterial strain used is by itself both ampicillin and tetracyclin sensitive. Therefore bacteria can only grow in medium with antibiotics when transformed with a plasmid containing intact ampicillin and tetracyclin resistance genes.
Here you see the result after the first step, which is transformation of the bacteria with the ligation mixture and the subsequent overnight growth on an agar plate supplemented with ampicillin. From these colonies recombinant clones have to be selected. Each of 24 randomly choosen colonies is used to inoculate corresponding wells in two multiwell plates and the cultures are grown overnight. The plates differ in the antibiotics they contain. The wells in this plate contains LB medium supplemented with only ampicillin while in this plate ampicillin and tetracyclin is added to the medium. The plates are incubated at 37°C overnight.

Now look at the result the next morning. All cultures were able to grow in the wells with ampicillin but some colonies were not able to grow on tetracyclin. Those wells very likely contain bacteria transformed with recombinant plasmids since they lost their tetracyclin resistance. So these are the potential candidates. They are transferred from the plate on the left to 1.5 ml tubes and can be processed directly in a miniprepping procedure to isolate and analyse the plasmids.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Capillairy Southern Blotting

This is the situation at the start of the blotting. The stack of paper towels is still dry, but starts to take up liquid from the gel, through the nylon sheet with the 2 paper sheets on top of it. Through the paper wick the sodium hydroxide solution is moving from the containers into the gel. The DNA fragments are denatured by the high pH and are carried with the moving solution towards the nylon sheet. This sheet acts as a filter (it has a high affinity for nucleic acids) and will not allow the DNA to pass.

 

This is the situation after overnight blotting. The towels have drawn up most of the sodium hydroxy solution from the containers and form a more compact stack now. The gel has become thinner, and the ethidium bromide has been washed out by the passing solution. The nylon sheet has single-stranded DNA bound to it's bottom side as a perfect copy of the agarose gel band pattern. The blotting set-up can be dismantled now, and the nylon sheet, the blot, can be used in a hybridization experiment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Random primer labelling of DNA fragments

This movie is about the random primer labeling of DNA fragments.

Here you see a drawing of one DNA fragment molecule. The two strands, shown in black and gray, are complementary to each other and in the opposite orientation.

The first step in the labeling procedure is the denaturation by boiling for a few minutes, after addition of an excess of randomprimers. Random primers are 6-nucleotides-long single stranded DNA molecules with a totally random sequence. Here they are drawn as gray dots.

After cooling down to room temperature, the primers, at least some of them, shown here, will find a complementary sequence on one of the strands. Since primers are excess, this happens faster than the reannealing of the two DNA strands which are also much longer.

The actual labeling reaction is started by the addition of a specific reaction buffer, the four dNTPs, of which one is labelled with for example Digoxigenin, and the Klenow DNA polymerase. The polymerase will elongate the annealed primers and thus synthesize new labelled DNA stretches, in the 5' to 3' direction, with the long single-stranded DNA strands as template. These new stretches are shown here in red.

After denaturation again by boiling the mixture can be used in a hybridization experiment, in which the labeled DNA stretches will be able to bind to sequences identical to the original DNA fragment.