Restriction Digests and Ligations Intro

In this lab, you'll work use restriction enzymes to cut DNA at specific sites, use ligase to rejoin some of the cut DNA fragments, and then use electrophoresis to see the results.

Restriction enzymes

Restriction enzymes are proteins that cut DNA at specific sites, determined by the nucleotide sequence of the DNA. Like most of the tools in a molecular biologist's toolkit, restriction enzymes are taken from cells. Cells do all sorts of tricks with DNA; learning how to manipulate DNA in a test tube usually means mimicking the way DNA is manipulated inside a cell. Cells manipulate DNA by using enzymes: proteins that catalyze specific chemical reactions. Molecular biologists get enzymes from cells, then use the enzymes in new ways.

Restriction enzymes cut DNA at specific sites. They recognize short sequences of nucleotides, called restriction sites, and cut the DNA at those sites. Restriction sites are usually four to eight base pairs long. There are many different restriction enzymes, each with its own specific restriction site. Restriction enzymes are also called endonucleases, because they cut nucleic acids somewhere in the midst of the molecule (endo- means in).

Restriction enzymes were the first DNA-altering enzymes to be isolated and used in the laboratory; in a sense, modern molecular biology began when a restriction enzyme was used to cut DNA in a test tube.

Why do cells have restriction enzymes?

Restriction enzymes cut up DNA. You might think that this would be bad for a cell; normally, cells don't cut up their own DNA. However, sometimes it's beneficial to cut up DNA that doesn't belong inside a cell. The restriction enzymes you'll use come from bacteria, and the bacteria use the enzymes to cut up DNA from viruses or other potentially dangerous sources. So the restriction enzymes function as a self-defense weapon for the cell.

How do the enzymes avoid cutting up the cell's own DNA? A bacterial cell may lack restriction sites that can be cut by its own enzymes, or it may chemically modify the DNA (by adding methyl groups) at the restriction sites.

Why is cutting DNA useful in the lab?

One key reason for cutting DNA in the lab is so you can join different DNA pieces together through ligation, forming recombinant DNA. That's the essence of gene cloning: create a new combination of DNA pieces, and then insert the recombinant DNA into cells.

Today's lab is intended to show you how to use enzymes to cut and ligate DNA. We also have a more practical purpose: making a molecular weight marker.

The restriction enzymes and DNA for this lab

In this lab, you'll use two different restriction enzymes to cut one kind of DNA. The restriction enzymes are:

EcoR1. ("Eco R one")Isolated from Escherichia coli. Cuts at this restriction site:

5'-GAATTC-3'

3'-CTTAAG-5'

HindIII. ("Hind three") Isolated from Haemophilus influenzae. Cuts at this restriction site:

5'-AAGCTT-3'

3'-TTCGAA-5'

The DNA is from a virus called lambda (abbreviated with the greek letter λ). it's a bacteriophage, meaning it's a virus that infects bacteria.

When you use electrophoresis to look at your results (next lab period), you should be able to see the difference between EcoR1-cut lambda DNA and HindIII-cut lambda DNA, because the enzymes cut the DNA at different places. The uncut DNA should give one big band on the gel, while the cut DNAs will show various smaller bands.

Sticky ends

Recall that restriction enzymes cut DNA at specific restriction sites. Since the two DNA strands are typically not cut at the same spot, an overhanging end is formed. If the DNA is cut by EcoRI, the cut ends will look like this (x represents any nucleotide outside the restriction site):

5'xxxxxxxxG AATTCxxxxxxxx3'

3'xxxxxxxxCTTAA Gxxxxxxxx5'

Note that the pieces both have identical single-stranded overhangs; these are called sticky ends. The two sticky-ended DNA fragments can stick to each other by base pairing: the As on one strand can hydrogen bond with the Ts on the other strand. As the two DNA fragments are temporarily held together by hydrogen bonds, ligase can re-form the covalent bond that was cut by the restriction enzyme.

Any two pieces of DNA that have been cut by EcoRI can stick together and be ligated. However, a piece that was cut with EcoRI won't stick to a piece that was cut by HindIII. Since HindIII's restriction site is different, it will leave different sticky ends:

5'xxxxxxxxA AGCTTxxxxxxxx3'

3'xxxxxxxxTTCGA Axxxxxxxx5'

If two fragments of DNA were cut with the same restriction enzyme, they will have the same sticky ends, and they could base-pair and hydrogen bond with one another. However, these hydrogen bonds are weak and easily broken. If you want to rejoin the DNA fragments more permanently, you'll need to use another enzyme to form covalent bonds joining the strands' sugar-phosphate backbones together. This process is called ligation.

Ligation

Ligation means joining pieces of DNA together; this process is catalyzed by the enzyme DNA ligase. Cells use ligase as a part of their normal DNA replication process. Cellular DNA is synthesized in fragments by DNA polymerase, and the fragments must be joined by ligase to make complete chromosomal DNA.

Ligase is also useful in molecular biology research. Cutting DNA into pieces, joining different pieces together in new combinations, and inserting the new recombinant DNA into cells is the basis of gene cloning. Cloning has been the foundation of much of the explosion of biological knowledge that has taken place in the last few decades.

Joining strands of DNA together means forming covalent bonds, and it requires energy. Some of the energy for ligation comes from breaking phosphoryl bonds on the DNA and some comes from additional ATP, which is present in the ligation buffer. The ligation buffer must always be kept cold to preserve the ATP.

Your experiment

You'll start with uncut lambda DNA. In the first part of the expriment, you'll attempt to cut some of this DNA in three different restriction digest reactions: some with Eco RI, some with Hind III, and some with both enzymes together. Then you'll take some of each of the three restriction digests and try to ligate the cut DNA fragments together. Later, you'll run a gel to see if the digests and ligations worked.

There are two experiments here: restriction digests and ligations. For each one, you need a control. For the restriction digest, the control will be the uncut DNA. If you see that the original DNA gives a single band on the gel, and the cut DNA gives multiple bands, then you know the digests worked. For the ligations, the cut DNA will be the control. If you see multiple bands in the cut DNA lane, and one large smear in the ligated DNA lane, then you know the ligations worked.

Molecular weight markers

For most molecular biology experiments, you'll eventually need to do electrophoresis to see your DNA or protein. Electrophoresis can separate molecules by size, but in order to determine the size of your unknown band on the gel, you'll need to compare them to bands of known size, called molecular weight markers. You need DNA molecular weight markers for DNA gels.

Today you can make a molecular weight marker by cutting lambda DNA with restriction enzymes. This works as a marker because the entire DNA sequence of lambda is known, and each restriction enzyme always cuts at the same sites, giving fragments of predictable size. In particular, lambda cut with HindIII is widely used as a marker, because it gives useful fragment sizes. You'll make some extra HindIII-cut lambda DNA to use as a marker in other labs later this quarter.