Introduction to Recombinant Genetics- Biology 350

Overview

"Restriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this "restriction" of viral growth stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these "restriction enzymes" to be purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific sites, generating discrete, gene-size fragments that could be re-joined in the laboratory. Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression." (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp)

Restriction Enzyme Types

"Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level there are many more than three different kinds.

Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.

Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Rather then forming a single family of related proteins, type II enzymes are a collection of unrelated proteins of many different sorts. Type II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors.

The most common type II enzymes are those like Hha I, Hind III and Not I that cleave DNA within their recognition sequences. Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize asymmetric DNA sequences because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoR I: GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200–350 amino acid range.

BOB by "Weird Al" Yankovic

The next most common type II enzymes, usually referred to as ‘type IIs" are those like Fok I and Alw I that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400–650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type IIs enzymes are much more active on DNA molecules that contain multiple recognition sites.

The third major kind of type II enzyme, more properly referred to as "type IV" are large, combination restriction-and-modification enzymes, 850–1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences; those that recognize continuous sequences (e.g., Eco57 I: CTGAAG) cleave on just one side; those that recognize discontinuous sequences (e.g., Bcg I: CGANNNNNNTGC) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied but their organization are consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.

Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. No laboratory uses have been devised for them, and none are available commercially." (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp)

Naming Restriction Enzymes

Restriction enzymes are named based on the bacteria in which they are isolated in the following example for the enzyme EcoRI:
    E      Escherichia (genus)
    co     coli (species)
    R      RY13 (strain)
    I       First identified Order ID'd in bacterium

Types of ends generated from digest

There are three types of ends generated from restriction digests, blunt ends (no overhangs), 5' overhangs, and 3' overhangs.

The same types of ends can be generated by different enzymes. These enzyme pairs are said to generate "compatible ends" because the overhangs can hydrogen bond with each other and base pair.

Frequency of enzyme recognition

The frequency of restriction enzyme recognition depends on the number of nucleotides used in the site recognition. Each position of the recognition site has four possible bases and therefore the probability of finding any one base is 1/4. To determine the probability of finding a particular sequence, you need to multiply the probability of each site by all of the other sites. If the length of the recognition site is 4 then the probability of finding that site is 1/4 * 1/4 * 1/4 * 1/4 = 1/256. For a site with six bases in the recognition site the probability would be (1/4)^6 = 1/4096. The reciprocal of these probabilties tell us that we would expect to see cuts in a completely random sequence, on the average, every 4096 bases for a six base recognition enzyme. Now, since most DNA sequences are not completely random, we do no always see a normal distribution of fragment lengths around this estimated size.

Cutting conditions

Most restriction enzymes function adequately at pH 7.4. All type II enzymes require Mg2+ and vary in their requirement for ionic strength (usually provided by NaCl). Proteins are further stabilized by addition of a strong reductant such as dithiothreitol (DTT). Some restriction enzymes are sentitive to protein dilution and are benefited by the addition of non-enzymatic proteins such as bovine serum albumin (BSA).

Restriction enzyme stocks are most often shipped with a 10X reaction buffer that is optimized for the enzyme. Most of these reaction buffers can be classified into four general categories, based on their ionic strength:

0 mM NaCl = low salt buffer (L)

50 mM NaCl = medium salt buffer (M)

100 mM NaCl = hi salt buffer (H)

150 mM NaCl = very high salt buffer (VH)

When digesting with two or more enzymes in the same buffer that do not use the same buffer, it is important to consult activity tables, such as 3.1.2, which list the relative activities in the various classes of restriction enzyme concentrations. For example, if a double digest was being made with EcoRI (hi salt) and HpaII (low salt, KCl), Table 3.1.2 tells us that both enzymes are active in medium salt. Since HpaII requires KCl instead of NaCl, you would use a medium concentration KCl buffer.

When some restriction enzymes find themselves in suboptimal conditions they cut abnomally. For example, EcoRI, when cutting in low salt buffers does not just recognize its normal sequence G^AAATTC but will also recognize ^AATT sites. This kind of activity is called *Star activity. You must therfore be careful of reaction conditions when dealing with enzymes that have known star activity.

Most restriction enzyme reactions can be run at 37°C. There are some enzymes, however, that have other optimal temperatures (see table 3.1.1).

Enzyme Reactions

By convention, enzyme activity is defined in terms of units, where 1 unit is the quantity of enzyme that will completely cut all sites on 1 µg of DNA in 1 hour. Since not all DNA sequences contain the same number of sites for an enzyme, the DNA that is used for definition of the unit scale must also be included. There are some common viral and plasmid DNAs that are commonly used to define the enzyme unit, such as lambda, SV40, and pBR322.

Restriction enzymes are shipped and stored in 50% glycerol to stabilize the protein and prevent freezing, which often denatures protein and inactivates enzymes. Enzyme function is inhibited by high concentrations of glycerol > 5% and so stock enzymes must be diluted at least 1/10 in reaction mixtures in order to prevent glycerol inhibition.

Since there is not a set number of enzyme sites per length of every DNA, when digesting a DNA for the first time it is a good idea to double the amount of enzyme and double the time of the digest in order to ensure complete digestion. The quality or purity of the DNA being digested can greatly impact its digestability. Restriction enzyme digests on DNA that is contaminated with proteins, excessive RNA, or base modifications can be hard to digest, requiring either longer digestion times, increased enzyme, or both.

Reduced digestion time and the dilution of enzyme can each be used to create partial digestions of target DNA. The best case senerio for a partial digest will produce a set of fragments that are randomly cleaved at each of the restriction enzyme sites. In reality, not all restrictions sites are cut with equal probability and so not all fragment size permutations may be represented at the same concentration.

Digestion of a large amount of DNA may be cost prohibitive at the 1U/1µg DNA concentration. You can use lower amounts of enzyme / DNA if the incubation times of the digest are extended. A good rule is to never use less than 0.5 units of enzyme in a digest. Many restriction enzymes become inactive when protein concentrations are reduced below this level. The dilution effect can sometimes be counteracted by adding BSA to keep overall protein concentrations high. Also, not all restriction enzymes are active for extended periods of time. Check the manufacturer data for stability information if you are planning extended incubations.

Restriction enzymes are stored at -20°C. When working with enzymes outside the freezer, make sure that you keep them on ice and return them to the freezer as soon as possible.

When setting up a restriction enzyme digest, always add the restriction enzyme last so that it has the proper buffer and approximate reaction conditions.

A typical restriction enzyme digest can be set up as follows:

Note that there is flexibility in the amount of DNA and the corresponding amount of water that you can add to the reaction. You should be warned, if you choose to add a large volume of a dilute DNA to the reaction and the DNA is contaminated with proteins or RNA, the reaction may be inhibited. Take care, when adding large volumes of DNA, to make sure that the DNA is pure.

Stopping Restriction enzyme digestions

After the incubation period is complete, digestions may be stopped by adding EDTA, which chelated Mg2+. Some enzymes may be inactivated by heating (see table 3.1.1) but others may require extraction with a protein denaturant such as phenol/chloroform or separation of the protein from the DNA on a column (Qiagen and others).

Separation of restriction enzyme fragments by length

DNA fragments that vary in size can be separated by electrophoresis in an agarose or polyacrylamide gel.

DNA bands in the gel can be visualized by adding ethidium bromide or other DNA binding dye such as SYBR green. Exposure of the gel to UV light will cause the bound dye to fluoresce and thus allow the DNA to be visualized and the relative positions photographed.

The size of the DNA fragments can be estimated by comparison to the relative migration of DNA standards of known size.

Note that the plot of migration vs size produces a curve. The curve can be linearized by plotting the distance migrated against the log(DNA size). A linear regression analysis on log transformed data can be used to determine the quality of the data and estimate the size and associated errors of unknown fragments.

DNA ligation

The cloning of DNA fragments into plasmid, phage, or other vectors requires the ligation of the DNA fragments to the vector. This requires the enzyme DNA Ligase.

T4 DNA ligase is isolated from the T4 bacteriophage and is commercially available. There are also several other commercially available sources of ligase which use ATP as an energy source. Ligase isolated from E.coli uses NAD as an energy source instead of ATP.

As illustrated above, the ligase enzymes require that a adjacent bases have a 5' phosphate and a 3' hydroxyl group to serve as substrates.

T4 DNA Ligase can ligate both blunt or sticky ended DNA.

The efficiency of ligation is much higher for sticky ends than for blunt ends. For blunt ends we need about 10X more ligase and lower temperatures (12-14°C, less thermally efficient than 20°C) in order to get ligation.

Exercise:

1. If you have a fragment, I, cut with EcoRI, that you want to insert into a linearized vector, V cut with EcoRI, and you mix equimolar amounts of each with ligase, what are all of the possible ligation products?

2. If you wanted to increase the percent of vector that contained insert, what would you change?

3. How does molar concentration impact self-ligation vs intramolecular ligation?

Addition of Linkers to blunt-ended DNA

If you have a fragment with blunt ends that you want to clone, you can improve the efficiency of ligation by adding short linkers that contain a restriction enzyme site. Usually the linker is present in a 100X molar concentration to the target fragment to ensure that all ends receive a linker. With this excess of linker, it is highly probable that more than one linker will be added. This is resolved by cleaving the excess linkers off with the restriction enzyme specific for the site that the linkers carry.

When selecting linkers, choose linkers with restriction enzyme sites that are not contained in the fragment to avoid cutting up your target.

Addition of adapters to blunt-ended DNA

An alternative, to avoid cutting up the target, is to use adapters. Adapters are designed to ligate at the blunt end but not at the sticky end (no 5' P).

Producing sticky ends with homopolymer tailing

The enzyme terminal deoxynucleotidyl transferase (or terminal transferase for short) adds nucleotides tothe 3' end of DNA without requiring a template.

If only a single nucleotide triphosphate is available to the enzyme, then it will add a homopolymer tail to the 3' ends of linear DNA. By adding complementary tails to the fragment to be inserted and the vector, a fragment of DNA can be inserted into a vector. The advantage of this system is that the vector will not ligate back together without an insert. Since the complementary homopolymer tails are most likely not the same length, the klenow fragment of DNA polymerase will need to be added along with the ligase in order to repair nicks before ligation.