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Restriction Endonucleases - molecular scissors for DNA(I)

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Restriction Endonucleases - molecular scissors for DNA(I)

Today, in the age of molecular biology, the study of a genomic DNA from any organism is a central point of the drive to understand biology.

Genomes are composed of large DNA pieces on the order of millions of base pairs, while a scientist could reasonable only handle fragments of DNA a few thousand base pairs long.




How to reproducibly cut the genomic DNA into fragments that are small enough to handle?

Restriction Endonucleases - molecular scissors for DNA(II)

A method for reproducibly cutting genomic DNA into controllable pieces was needed to improve the study of genomes forward.

This question was first posed in the 1970s.

Before the 1970s, it was a relatively simple to isolate  and purify DNA, and then randomly cut it up using chemical or mechanical means.

The random cutting was an unacceptable way to get smaller fragments of DNA, since it was not possible to see the original order of these pieces.

The specific order of DNA fragments is essential to its function.

Restriction Endonucleases - molecular scissors for DNA(III)

As with other very important discoveries in biology the bacteriology provided the step forward.

The step forward was the discovery of a specific bacterial restriction and modification system able to cut and/or modify DNA in a test tube.

This system acts as a kind of immune system, protecting the bacteria from the invasion of foreign DNA (e.g., bacteriophages).

Any restriction and modification system reside in 2 types of enzymes: endonucleases and methylases.

Because endonucleases cut double stranded DNA at restricted sites, they are named restriction endonucleases or restriction enzymes.

Restriction Endonucleases - molecular scissors for DNA(IV)

The restriction endonucleases provided molecular biologist with a tool to manipulate DNA and to obtain constantly sized DNA fragments.

The restriction endonucleases are named after the Genus and species  from which they are isolated.

The restriction endonucleases are used for a wide range of applications, including cloning, Southern hybridization analysis, DNA sequencing and global

gene expression analysis.

Recombinant DNA technology, essential for the field of molecular biotechnology, is unlikely to have been developed without the discovery of restriction endonucleases.

Restriction Endonucleases - molecular scissors for DNA(V)

Restriction endonucleases were discovered during experiments to determine the ability of a bacteriophage to infect B and K laboratory strains of Escherichia coli.

After the incubation with the different strains of E. coli, the ability of the bacteriophage to kill infected cells was monitored.

After infection, the bacteriophage will replicate and normally kill infected bacteria releasing ~100 bacteriophages/cell.

Restriction Endonucleases - molecular scissors for DNA(VI)

When bacteriophage spilling out of E. coli B cells were isolated, they were found to be very successful at re-infecting E. coli strain B cells.

But when these bacteriophages were incubated with E. coli K, it was found that only a few of them could manage to replicate.

After a series of incubations of the these bacteriophages with E. coli strain K, their ability to infect and kill E. coli K was increased.

With a switch back to E. coli strain B, these bacteriophages (now able to infect strain K to a high degree), showed a reduction of their ability to infect.

Restriction Endonucleases - molecular scissors for DNA(VII)

The results of this experiment suggested two things:

The first was that strain K and strain B each had some mechanism that 'restricted' the infective ability of bacteriophage isolated from the other strain.

This was supported by the reduction of infectivity of the bacteriophage when it was initially switched from one strain to the other.

The second was that the restriction of the bacteriophage's infectivity was lost as the bacteriophage's DNA was replicated inside the bacterial cell.

This was evidenced by the return of the bacteriophage's infectivity after several passages inside of the new host strain of bacteria.

Restriction Endonucleases - molecular scissors for DNA(VIII)

What is this restrictive mechanism?

It turns out that a bacterium 'labels' its own DNA by “modifying” it. For example, in some bacteria the cytosine has an extra methyl group.

This modification is referred to as methylation. Any methylation is “conserved” in newly synthesized DNA.

When a foreign DNA comes inside of the bacterial cell, as when a bacteriophage injects its DNA into a bacterium, it does not have these modifications and thus is recognized as unknown to the cell and rapidly destroyed.

To perform this function a series of enzymes evolved to recognize and cut not methylated DNA. These enzymes were later named restriction endonucleases.

Restriction Endonucleases - molecular scissors for DNA(IX)

Restriction and Modification System.The existence of this system explains the differences in the infection rate between E. coli B and K strains above.

If the bacteriophage DNA is successful in avoiding the destruction, its replication inside the bacterium over time would mark it with the same modifications as the host. The modified bacteriophage DNA would then no longer be recognized as foreign and be able to infect a bacterium at a much greater frequency.

Discovery of this relatively simple system able to make a distinction between host and foreign DNA provided biologist with a new molecular tool: bacterial enzymes that could cut DNA, just like scissors cut paper.

How Restriction Enzymes Work? (I)

The bacterial restriction and modification system is analogous to an immune system.

Restriction and modification system combines two enzyme activities: a modification methyltransferase and a restriction endonuclease.

The methyltransferase transfers methyl groups from S-adenosylmethionine to specific bases (cytosine or/and adenosine) within the recognition sequence, generally one in each strand.

If one strand is already methylated, the second strand remains a substrate.

The biochemical activity of a restriction endonuclease is the hydrolysis ('digestion') of the phosphodiester backbone at specific sites in a DNA sequence.

The restriction endonuclease cleaves the DNA but only if the recognition site is not methylated in either strand.

How Restriction Enzymes Work? (II)

In almost all cases, a bacterium that makes a particular restriction endonuclease also synthesizes a companion DNA methyltransferase, which methylates the DNA target sequence for that restriction enzyme, thereby protecting it from cleavage.

It has been mentioned that restriction endonucleases recognize and cut foreign DNA but the way these enzymes cut DNA is varied.

Some cut the foreign DNA randomly.

While others recognize a particular DNA recognition sequence and then either cut within this DNA sequence or several nucleotides away from it.

Where a restriction endonuclease cuts within a DNA molecule is one easy way for its classification.

How Restriction Enzymes Work? (III)

The restriction and modification (R/M) systems are classified into three types: I, II and III.

For type I recognition of DNA sequence and both enzyme activities are all carried out by a single oligomeric protein.

The type I proteins contain three different activities:

- the first is to recognize the DNA sequence;

- the second is to methylate the DNA at the recognition

site;

- the third is to cut the DNA.

The DNA is cut at random positions, often several kbp away from the  recognition sequence.

The cofactor for both activities (methyltransferase and endonuclease) is S-adenosyl methionine.

The endonuclease activity also needs both Mg2+ and ATP.

How Restriction Enzymes Work? (IV)

Type III R/M systems

Type III R/M systems, like type I, use a single oligomeric protein for both activities but they contain only two sorts of activities:

- one to recognize the target sequence on the DNA and carry out the methyl transfer reaction;

- a second to cut the DNA.



Type III enzymes cut the DNA 25-27 bp away from the recognition site.

How Restriction Enzymes Work? (V)

Type II R/M systems

In type II R/M systems, both activities are carried out by separate proteins which independently recognize the same DNA sequence.

Type II modification enzymes are generally monomeric proteins and their restriction enzymes are usually dimmers of identical subunits.

Within each type II R/M system, there is no homology  between the restriction and modification enzymes.

The modification enzymes from different type II R/M systems are often homologous to each other, but the endonucleases are a diverse group of proteins.

How Restriction Enzymes Work? (VI)

Type II R/M systems

By convention, restriction enzymes are named after their host of origin. For example, EcoRI was isolated from Escherichia coli (strain RY13), HindII and HindIII from Haemophilus influenzae, and XhoI from Xanthomonas holcicola.

In contrast to type I R/M systems, type II endonucleases require only Mg2+ as a cofactor for DNA cleavage, and they cut the DNA at specific phosphdiester bonds within (or nearest to) the recognition site.

The structure of two type II endonucleases, EcoRI and EcoRV, have been determined by X-ray cristallography.

The two structures have little in common and the way in which they interact with their recognition sequences is totally different.

How Restriction Enzymes Work? (VII)

Type II R/M systems

In both cases, the parts of the enzymes that interact with the DNA have structures that are unlike any of standard protein motifs for DNA recognition.

However, these structural elements still contact the bases in the target sequence by networks of hydrogen bonds.

For EcoRI, these interactions improve enzyme binding more strongly to its recogniton site than to anywhere else on the DNA, while part of the interaction energy is used to deform the structure of the DNA prior to catalysis.

EcoRI - DNA Complex Structure

The DNA in the complex is substantially unwound in the centre of the recognition sequence by the insertion of the

ends of two helices, one from each monomer, into the major groove.

Loop structures from each monomer run along the major groove towards the end of the recognition sequence and interact with the phosphate backbone at the point of cleavage.

How Restriction Enzymes Work? (VIII)

Type II R/M systems

EcoRV, also isolated from an E.coli strain, recognizes the sequence :  5' - GATATC - 3‘ 3' - CTATAG - 5‘ but unlike EcoRI,

cleaves between the central T:A → blunt-ended products.

In contrast, the EcoRV endonuclease binds with equal affinity any DNA sequence, including the recognition site, but the cuting activity is only present at the level of specific complex EcoRV/DNA.

At the level of this specific complex (EcoRV/DNA), the   DNA sequence corresponding to the recognition site is severely distorted, while in any nonspecific complex DNA has a normal structure.

Only this specific complex (EcoRV/DNA) is able to bind the Mg2+ ions needed for catalytic reaction.

EcoRV - DNA Complex Structure

The structure of EcoRV bound to a substrate oligonucleotide shows a remarkable degree of distortion in the DNA, which is substantially bent towards the enzyme at the minor groove.

Loops from each monomer wrap around the front of the DNA into the major groove and make specific contacts with the recognition sequence. The enzyme is

only able to achieve this distortion when interacting with its target sequence.

How Restriction Enzymes Work? (IX)

Type II R/M systems

PvuII endonuclease isolated from Proteus vulgaris cut the sequence:  5’-CAGCTG-3’

3’-GTCGAC-5’

between the two central G:C base pairs.

PvuII - DNA Complex Structure

Like EcoRV, PvuII binds the DNA with the minor groove towards the enzyme, and interacts with the target sequence in the major groove via a beta-ribbon which wraps around the side of the DNA.

However, unlike EcoRV there is no apparent need for distortion in the recognition process and the bound DNA in the complex is essentially undistorted B-form.

How Restriction Enzymes Work? (X)

Type II R/M systems

Type II endonucleases have extensive applications in the field of molecular biology, in the DNA dissection and in the recombinant DNA technology.

Without restriction endonucleases the remarkable developments in molecular genetics, biochemistry and cell biology in the second half of the XX-th century could not have taken place.

The specificity of type II restriction endonucleases for their recognition sites on DNA is the key point for their ability to distinguish between the recognition sequence and any other DNA sequence.

How Restriction Enzymes Work? (XI)

Type II R/M systems

Type II restriction enzymes have become an exceptional tool for the molecular biologist to specifically cut DNA.

These endonucleases bind to DNA at any position and then travel along the strand of DNA until they reach a recognition sequence.

The recognition sites provide the key to how one DNA fragment can be cut into two in a specific manner.

The recognition site vary in size; for some endonucleases recognizing sequence motifs in DNA are 4 bp long, while others recognize sequences that are 20 bp long.

How Restriction Enzymes Work? (XII)

Type II R/M systems

The nucleotide pattern that is recognized by different restriction enzymes is variable palindromic sequence.

A palindromic sequence is the same when read in 5' to 3‘ direction on either complementary strand of DNA:

5’-GAATTC-3’

3’-CTTAAG-5’

EcoRI recognizes a six-nucleotide pattern that reads GAATTC from the 5' to the 3' end of the DNA molecule.

The complement of this sequence (on opposite DNA strand) also is GAATTC when read from 5' to 3'.

The DNA cleavage by EcoRI is symmetrical (between the G and the A when reading 5' to 3') on both strands.

How Restriction Enzymes Work? (XIII)

An important characteristic of this DNA cleavage is the 'overhanging” or “sticky” ends of the resulted molecule.

This feature is often used in the process of DNA cloning to help the adhesion of DNA insert to linear vector.

Overall, restriction endonucleases are quite variable in the DNA ends that they produce upon cutting, for example leaving a 5’ overhang (as in EcoRI), a 3‘ overhang, or no overhang at all ('blunt' ends).

Restriction Enzyme Recognition Sequences (I)

The length of restriction recognition sites varies.

The enzymes EcoRI, SacI and SstI each recognize a 6 bp sequence of DNA, whereas NotI recognizes a sequence 8 pb in length, and the recognition site for Sau3AI is only 4 pb in length (“four cutter”).

Length of the recognition site dictates how frequently the enzyme will cut in a random sequence of DNA.

A quick calculation and a couple of basic assumptions are enough to estimate how frequently it should cut a piece of DNA.

For example, with the four nucleotide bases types that make up DNA the probability of any one nucleotide occurring at a given location is ¼.

In the case of a 'four cutter' a specific sequence of four nucleotides must be present and assuming that each nucleotide has an equal chance (i.e. ¼) of occurring at any particular site, then ¼ x ¼ x ¼ x ¼ = 1/256, a “four-cutter” should on average cut once every 256 base pairs.

Restriction Enzyme Recognition Sequences (II)

A similar calculation can be applied to any restriction enzyme as long as the size of its recognition site is known, making it possible to predict the size and number of a DNA fragments that would be obtained by cutting a DNA molecule of known size.

For example, enzymes with a 6 bp recognition site will cut, on average, every 46 or 4096 bp.

This fact gave molecular biologists the method they required to produce DNA fragments of known size for their experiments, as in gene mapping and genetic

engineering.

Restriction Enzyme Recognition Sequences (III)

Isoschizomers

Different restriction endonucleases can have the same recognition site - such enzymes are called isoschizomers For example, the recognition sites for SacI and SstI are identical: 5’-GAGCT↓C-3’ (3’ overhang).

In some cases isoschizomers cut identically within their recognition site, but sometimes they do not.

Isoschizomers often have different optimum reaction conditions, stabilities and costs, which may influence the decision of which to purchase.



Restriction Enzyme Recognition Sequences (IV)

Restriction recognitions sites can be unambiguous or ambiguous.

For example, BamHI recognizes only the sequence:

5’-GGATCC-3’. This endonuclease has an unambiguous restriction recognition site.

In contrast, HinfI recognizes a 5 bp sequence:

5’-GANTC-3’, ('N' stands for any nucleotide). HinfI has an ambiguous recognition site.

XhoII also has an ambiguous 6 bp recognition site:

5’-PuGATCPy-3’ [Py stands for pyrimidine (T or C) and Pu for purine (A or G)], so XhoII will recognize and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC.

Restriction Enzyme Recognition Sequences (V)

The recognition site for one enzyme may contain the restriction site for another.

For example, note that a BamHI recognition sequence (5’-G↓GATCC-3’) contains the recognition site for Sau3AI (5’-N↓GATCN-3’).

Consequently, all BamHI sites will cut with Sau3AI.

Similarly, one of the four possible XhoII sequences will also be a recognition site for BamHI and all four will cut with Sau3AI.

Restriction Endonuclease Activity (I)

Endonuclease enzymatic activity depends of:

1. Buffer composition,

2. Incubation temperature,

3. DNA methylation,

4. DNA substrate.

Restriction Endonuclease Activity (II)

Buffer Composition

Different restriction endonucleases have differing preferences for ionic strength (salt concentration) and major cation (sodium or potassium).

A set of 4 to 5 different buffers will “cover up” a large number of available enzymes, although there are a few that require a unique or specific buffer environment.

In all cases, a major function of the buffer is to maintain pH of the reaction (usually at 8.0).

Additionally, some enzymes are more selective about having their optimal buffer than other enzymes.

Obviously, use of the wrong buffer can lead to poor cleavage efficacy.

Restriction Endonuclease Activity (III)

Incubation Temperature

Most restriction endonucleases cut best at 37°C, but there are many exceptions.

Endonucleases isolated from thermophilic bacteria cut best at temperatures ranging from 50 to 65°C.

Some other endonucleases have a very short half life at 37°C and for them it is recommended incubation at 25°C.

Restriction Endonuclease Activity (IV)

DNA Methylation

Almost all E. coli strains used for propagating cloned DNA contain two site-specific DNA methylases:

1. Dam methylase adds a methyl group to the adenine in the sequence 5’-GATC-3’, yielding a sequence symbolized as 5’-GmATC-3’.

2. Dcm methylase adds a methyl group to the internal cytosine in 5’- C(A/T)GG-3’, generating the sequence

5’-CmC(A/T)GG-3’. The practical importance of this phenomenon is that a

number of restriction enzymes will not cut methylated DNA.

Restriction Endonuclease Activity (V)

DNA Methylation

Two examples relative to Dam methylation should illustrate this concept.

MboI and Sau3AI are isoschizomers that recognize and cut the sequence GATC, which is accurately the sequence recognized by Dam methylase.

Digestion of GmATC by MboI is completely inhibited, while digestion by Sau3AI is unaffected by methylation.

Restriction Endonuclease Activity (VI)

DNA Methylation

The recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase.

In spite of this, if that sequence is followed by a C or preceded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited.

5’-GATCGAT-3’ → 5’-GmATCGAT-3’

5’-ATCGATC-3’ → 5’-ATCGmATC-3’

Thus, a random sequence of DNA propagated in most strains of E. coli, half of the ClaI recognition sites will not cleave.

Restriction Endonuclease Activity (VII)

DNA Substrate

The cutting efficiency of a restriction endonuclease for the same recognition sequence at different locations in a piece of DNA can vary 10 to 50-fold.

This is apparently due to the influences of nucleotide sequences bordering the recognition site, which possibly can either enhance or inhibit enzyme binding or activity.

A related situation is seen when restriction recognition sites are located at or very close to the ends of linear fragments of DNA.

Most enzymes require a few bases on either side of their recognition sequence in order to bind and cleave.

Many of the companies that sell enzymes provide in their catalogs the 'end requirements' for a variety of enzymes.

Restriction Endonuclease Activity (VIII)

Star Activity

When DNA is digested with certain restriction enzymes under non-standard conditions, cleavage can occur at sites different from the normal recognition sequence – such anomalous cutting is called 'star activity'.

For example, EcoRI can exhibit star activity; in this case, cutting can occur within a number of sequences that differ from the normal recognition site 5’- GAATTC -3’ by a single base substitutions.

Restriction Endonuclease Activity (IX)

Star Activity

Non-standard conditions that may induce star activity take account of:

1. High pH (>8.0) or low ionic strength (e.g. if you forget to add the buffer).

2. Glycerol concentrations > 5% (enzymes are usually sold as concentrates in 50% glycerol).

3. Extremely high concentration of restriction endonuclease (>100 U/ g of DNA).

4. Presence of organic solvents in the reaction mixture (e.g. ethanol, DMSO).

Restriction Endonuclease Activity (X)

Digestion with Multiple Endonucleases

Digesting DNA with two endonucleases having different buffer requirements is relatively common. There are at least three approaches to control this situation.

Digest with both enzymes in the same buffer.

In many cases, even those a given buffer is not optimal for an enzyme, you can still get quite good cleavage rates.

Manufacturer catalogs usually contain a reference table recommending the best single buffer for conducting specific double digests.

Restriction Endonuclease Activity (XI)

Digestion with Multiple Endonucleases

Digesting DNA with two endonucleases having different buffer requirements is relatively common. There are at least three approaches to control this situation.

Cut with one enzyme, then alter the buffer composition and cut with the second enzyme.

This usually applies to situations where one enzyme like a low salt buffer and the other a high salt buffer.

In this case you can digest with the first enzyme for a time, add a calculated amount of concentrated NaCl, and cut with the second enzyme.



Restriction Endonuclease Activity (XII)

Digestion with Multiple Endonucleases

Digesting DNA with two endonucleases having different buffer requirements is relatively common. There are at least three approaches to control this situation.

Change buffers between digestion with two enzymes.

In some cases, two endonucleases will have totally incompatible buffers.

In that case, perform one digestion, recover the DNA (usually by precipitation) and resuspend it in the suitable buffer for the second enzyme.

Useful Information for a First-Time User(I)

At least three items deserve highlighting regarding restriction digestions:

1. Restriction endonucleases are expensive (some much more than others).

2. Restriction enzymes are, in general, heat labile.

Enzymes should be kept at -20°C except for brief periods of time on ice or in a small freezer box.

It is distressingly easy to leave a little box of enzymes on the lab bench overnight and lose several hundred dollars.

3. Contaminating one enzyme with small quantities of another can cause massive confusion and loss of time to yourself and your coworkers.

Never pipet an enzyme with anything other than a new pipet tip!

Useful Information for a First-Time User(II)

Setting up a simple restriction digestion is easy; there are three mandatory ingredients that need to end up in the same tube:

1. DNA - Reliable cleavage by restriction endonucleases requires DNA that is free from contaminants such as phenol or ethanol.

Excessive salt will also interfere with digestion by many enzymes, while some are more tolerant of that problem.

2. An appropriate buffer - Different endonucleases cut optimally in different buffer systems, due to differing preferences for ionic strength and major cation.

When you purchase an enzyme, the company invariably sends along the matching buffer as a 10X concentrate.

3. The restriction enzyme! Do not laugh - at some point in your career, you will set up a group of digests, forget to add the enzyme, and wonder why nothing cut.

Useful Information for a First-Time User(III)

The amount of DNA to be used depends on the task. For example, consider a typical diagnostic digestion to confirm the identity of a plasmid.

1. In this case, 1 g of DNA should suffice and 20 l would be a convenient volume for the reaction mixture.

2. After deciding which enzyme to use, look for buffer and incubation temperature.

3. Into an Eppendorf tube pipet 2 l of 10X buffer, the DNA and water to bring the total volume to 20 l.

4. Finally, add 1-2 units of restriction enzyme - most are provided at a concentration of 10 to 20 units per l.

5. It is important to shakeup the reaction mixture, by 'flicking' the tube and then quaking down its contents.

6. After incubation at the recommended temperature for 60-90 minutes, analyze the products by gel electrophoresis.

CURS 10

OTHER ENZYMES

Nucleases: DNase and RNase

I

Most of the time nucleases are the enemy of the molecular biologist who is trying to preserve the integrity of RNA or DNA samples.

On the other hand, deoxyribonucleases (DNases) and ribonucleases (RNases) have certain indispensible roles in molecular biology laboratories.

Numerous types of DNase and RNase have been isolated and characterized.

They differ among other things in substrate specificity, cofactor requirements, and whether they cleave nucleic acids internally (endonucleases), chew up from the ends (exonucleases) or attack in both of these modes.

In many cases, the substrate specificity of a nuclease depends upon the concentration of enzyme used in the reaction, with high concentrations promoting less specific cleavages.

II

The most widely used nucleases are DNase I and RNase.

A, both of which are purified from bovine pancreas:

Deoxyribonuclease I cleaves double-stranded or single stranded DNA.

Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is therefore an endonuclease.

Major products are 5'-phosphorylated di-, tri- and tetranucleotides.

III

In the presence of magnesium ions, DNase I hydrolyzeseach strand of duplex DNA independently, generating random cleavages.

In the presence of manganese ions, DNase I cleaves both strands of DNA at approximately the same site, producing blunt ends or fragments with 1-2 base overhangs.

DNase I does not cleave RNA, but crude preparations of the enzyme are contaminated with RNase A; RNase-free DNase I is readily available.

Some of the common applications of DNase I are:

1. Eliminating DNA (e.g. plasmid) from preparations of RNA.

2. Analyzing DNA-protein interactions via DNase footprinting.

3. Nicking DNA prior to radiolabeling by nick translation

IV

Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA at the 3' end of pyrimidine residues RNase A degrades the RNA into 3'-phosphorylated

Some of the major use of RNase A are:

1. Eliminating or reducing RNA contamination in preparations of plasmid DNA.

2. Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the

cleavage products can be analyzed.

mononucleotides and oligonucleotides.

V

A number of other nucleases that are used to manipulate DNA and RNA are:

Exonuclease III from E. coli, used most commonly to prepare a set of nested deletions of the termini of linear DNA fragments.

Mung Bean Nuclease from Mung bean sprouts, used to remove single-stranded extensions from DNA to produce blunt ends.

Nuclease BAL 31 from Alteromonas, used for shortening fragments of DNA at both ends.

Nuclease S1 from Aspergillus, used commonly to analyze the structure of DNA:RNA hybrids (S1 nuclease mapping), and to remove single-stranded extensions from DNA to produce blunt ends.

Ribonuclease T1 from Aspergillus, used to remove unannealed regions of RNA from DNA:RNA hybrids

Alkaline Phosphatase

I

Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins.

This enzyme is most active at alkaline pH.

There are several sources of alkaline phosphatase that differ in how easily they can be inactivated:

Bacterial alkaline phosphatase (BAP) is the most active of the enzymes, but also the most difficult to destroy at the end of the dephosphorylation reaction.

Calf intestinal alkaline phosphatase (CIP) is purified from bovine intestine. This phosphatase is most widely used in molecular biology laboratories because, although less active than BAP, it can be effectively destroyed by protease digestion or heat (75°C for 10 minutes in the presence of 5 mM EDTA).

Shrimp alkaline phosphatase is derived from a cold-water shrimp and is successfully destroyed by heat (65°C for 15 minutes).






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