DNA ligase

Enzyme connecting two DNA fragments

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DNA Ligase It is an important enzyme in organism, which catalyzes reactions in DNA Replication and Repair process Plays an important role in. DNA ligases are divided into two categories: one is to use ATP energy to catalyze two Nucleotide Chain Interformation Phosphodiester bond Dependencies on ATP DNA ligases, and the other is to use Nicotinamide adenine dinucleotide ( NAD +) catalyzes the formation of phosphodiester bonds between two nucleotide chains.

DNA ligase is also called DNA binding enzyme molecular biology It plays a special and key role in connecting the DNA chain with a base 3 '- OH The terminal and the 5 '- P terminal of its adjacent base make them generate Phosphodiester bond To connect two adjacent bases. Ligase Catalysis Consumption required ATP 。

Chinese name: DNA ligase
Foreign name: DNA Ligase
Alias: DNA ligase

Role: Connecting the 3 '- OH end of the DNA strand
Principle: Catalysis
Conditions: ATP consumption required

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seven Connection means
eight DNA Polymerase

brief introduction

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DNA ligase

DNA Ligase, also known as DNA binding enzyme molecular biology It plays a special and key role in the process of synthesizing two DNA strands into one. Whether it is the binding of double strands or single strands of DNA, DNA binding enzymes can be formed by phosphate The bond connects the tail end of DNA at the 3 'end and the front end of DNA at the 5' end. Although there are other protein , such as DNA Polymerase With one strand of DNA as the template, pass the broken end of the single strand of DNA on the other side through polymerization Process formation of phosphoric acid DNA ligase stitches DNA fragments together to restore Restriction enzyme Two cut nucleotide Phosphoric acid double fat bond). however DNA Polymerase However, the binding process of DNA is just an incidental function of polymerization. The real work of playing the role of DNA binding reaction in cells is mainly DNA binding enzyme. The function of DNA binding enzyme is to bind broken DNA DNA replication And DNA repair DNA binding enzyme plays an important role in the above two mechanisms. With the development of molecular biology, almost all molecules biology laboratory Will use DNA binding enzyme Recombinant DNA Maybe this can also be classified as another important function.

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DNA ligase was found in three laboratories in 1967 At the same time Of, originally in Escherichia coli Found in cells. It is a kind of enzyme that closes the gap on the DNA strand ATP or NAD The energy provided by hydrolysis catalyzes the formation of 5 '- PO4 of a DNA strand and 3' - OH of another DNA strand Phosphodiester bond 。 But these two chains must be the same Complementary chain Paired（ T4DNA ligase Except), and it must be two adjacent DNA strands to be catalyzed by DNA ligase into phosphodiester bond.

nature

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The DNA ligase of Escherichia coli is a 75Ku Polypeptide chain 。 yes Trypsin It is sensitive and can be hydrolyzed. The small fragments formed after hydrolysis still have some activity and can catalyze the enzyme and NAD (instead of ATP) reaction to form enzyme AMP intermediate, but AMP cannot be transferred to DNA to promote the formation of phosphodiester bond. DNA ligase has about 300 molecules in Escherichia coli cells, and DNA Polymerase I's Molecular number This is also a reasonable phenomenon. Because the main function of DNA ligase is to Catalytic polymerization , Fill Double stranded DNA The gap on the single strand of DNA is closed after the gap on the double strand of DNA. This is DNA replication , repair and recombination, ligase defective Mutant DNA replication, repair and recombination cannot be carried out.

Bacteriophage T4 DNA ligase molecule is also a Polypeptide chain Its molecular weight is 60Ku, and its activity is easily 0.2 mol/L KCl and Spermine Inhibited. The catalytic process of this enzyme requires ATP assistance. T4DNA ligase can Connect DNA -DNA，DNA- RNA , RNA RNA and double stranded DNA Viscous end or Flat end 。 In addition, NH4C1 can be improved in Escherichia coli The catalytic rate of DNA ligase is not effective for T4DNA ligase. Neither T4DNA ligase nor Escherichia coli DNA ligase can catalyze the connection of two free DNA chains.

purpose

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DNA connection

DNA ligase is mainly used for genetic engineering and will be Restriction endonuclease The sticky ends "cut" are reassembled, so it is also called "gene needle and thread".

People's Education Edition High School Biology Elective E · coli DNA ligase mentioned in 3, derived from Escherichia coli , can be used to connect Viscous end ； T4DNA ligase , from T4 bacteriophage , can be used to connect sticky ends and flat ends, but the connection efficiency is low.

classification

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T4DNA ligase

T4 DNA Ligase is an ATP dependent DNA ligase that catalyzes two DNA strands Double chain Upper adjacent 5 ' phosphoric acid Formation between radical and 3 ′ hydroxy group Phosphodiester bond 。 Double stranded DNA Flat end , compatible adhesive end and single chain notch therein molecular biology It has a wide range of applications. T4DNA ligase is Prokaryotic expression ， column chromatography Purified high-purity T4DNA enzyme, SDS-PAGE It is displayed as a 62kD protein band. This product comes with 10xLigationBuffer containing ATP. It is suitable for DNA fragment splicing, cloning and other reactions.

Content and storage method

Name: T4DNALigease Quantity: 20ml Storage condition: - 20 ℃

DNA connection

Name: 10xLigationBuffer Quantity: 100ml Storage condition: - 20 ℃ T4DNA connection enzymatic activity Is 3u/ml, and the unit is defined as Weiss unit (0.01 Weiss unit activity of T4DNA graft can be connected with 95% 1g λ DNA HindIII enzyme digest when reacting in 20ml system at 16 ℃ for 20 minutes). No DNA Exonuclease or Endonuclease Activity. More than 50 routine DNA reactions can be performed. Low temperature transportation , stored at - 20 ℃. The validity period is 6 months.

10 × LigationBuffer Composition:

Tris-HCl pH7.8600mM

MgCl215mM

DTT100mM

ATP10mM

Connection conditions

It is recommended to carry out the reaction in 10~20ml reaction system, and the maximum concentration of DNA in the reaction can reach 1mM (1kbDNA is about 1mg/ml). First mix the DNA fragments to be connected, add 10 × LigationBuffer to the final concentration of 1 ×, and then add an appropriate amount of T4DNALigease Evenly mix 。 The optimum connection temperature is 16 ℃. The adhesive end bonding efficiency is high, adding 0.2~1ml T4DNA Ligase can complete the reaction at room temperature or 16 ℃ for 1~3 hours; Flat end The connection efficiency is low. When 1ml T4DNA Igase is added, the reaction usually needs to be completed overnight at 16 ℃ or 4 ℃.

matters needing attention

1) 10xLigationBuffer contains ATP, which should be placed at room temperature to melt or palm temperature assisted melting before use, and then placed on ice. Do not heat to melt to avoid ATP degradation.

2) T4DNA Igase is heat sensitive and easy to Inactivation 。 Please put it on ice when using, and put it in the refrigerator immediately after use Cryopreservation 。

3) If it is necessary to inactivate T4DNALigase after reaction, it can be incubated at 65 ℃ for 10min.

Thermal stability DNA ligase

Thermostable DNA Ligase (thermostableDNAligas), from thermophilic Thermophilic actinomycetes (Thermoactinomyces thermophilus) Separation and purification One can catalyze two Oligonucleotide probe One that is connected Nuclease 。 It has the activity of ligase at 85 ℃, and still maintains the activity of ligase after repeatedly warming to 94 ℃. In gene Cloning experiment Plays an important role in.

function

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Restriction endonuclease (hereinafter referred to as restriction enzyme): restriction enzyme mainly exists in microorganisms (bacteria mould Etc.). One restriction enzyme can only recognize one specific nucleotide sequence , and can be used in specific Tangency point Upper cut DNA molecule 。 yes Specificity To cut the DNA strand Phosphodiester bond Of Nuclease （“ Molecular scalpel ”）。 Found in prokaryotic organisms, more than 100 kinds of prokaryote All contain this enzyme. yes Recombinant DNA technology and gene diagnosis An important class of tool enzyme 。 For example, from Escherichia coli A restriction enzyme found in can only recognize GAATTC sequence and cut this sequence between G and A. More than 200 restriction enzymes have been found with different cut points. Bacillus thuringiensis The insect resistance gene in can be cut down by some restriction enzyme. stay genetic engineering Play a role in.

DNA Ligase : Mainly connected between DNA fragments Phosphodiester bond , play a role in connection and genetic engineering. In genetic engineering, Escherichia coli Ligase only connects Viscous end T4 ligase can connect both the sticky end and the flat end.

DNA Polymerase : Mainly connect DNA fragments with single Deoxynucleotide The phosphodiester bond between the two plays a role in DNA replication.

DNA Polymerase Only a single nucleotide can be added to the hydroxyl group at the 3 'end of the existing nucleic acid fragment to form a phosphodiester bond; DNA ligase forms phosphodiester bonds between two DNA fragments, not between a single nucleotide and DNA fragments.

DNA connection

DNA polymerase uses a DNA chain as a template to pass a single nucleotide through Phosphodiester bond Form a line with Template chain Complementary DNA strand; DNA ligase Double chain The two notches on the are connected at the same time. Therefore, DNA ligases do not require templates. RNA polymerase (also known as RNA replicase RNA synthetase) catalytic activity RNA polymerase takes the complete double stranded DNA as the template, and the double stranded structure of DNA is partially uncoupled during transcription, and the double stranded structure of DNA remains after transcription. Eukaryote RNA polymerase: the transcription mechanism of eukaryotes is much more complex, with three types nucleus RNA polymerase in: RNA Polymerase I Transcription rRNA ， RNA Polymerase II Transcription mRNA ， RNA Polymerase III Transcription tRNA And others Small RNA 。 stay RNA replication And transcription.

Reverse transcriptase ： RNA Guided DNA Polymerase , there are three enzymatic activity RNA directed DNA polymerase, RNA enzyme , DNA directed DNA polymerase. stay Molecular Biology Technology As an important tool enzyme, it is widely used to establish Gene library , access Target gene And so on. It plays a role in genetic engineering.

Helicase : is a kind of enzyme that unlocks the hydrogen bond ATP To supply energy. They often rely on the existence of a single chain and can identify Copy Fork Single chain structure of. There are many similar helicases in bacteria, all of which have ATPase Activity. Most of the moving directions are 5 '→ 3', but there are also cases where 3 '→ 5' moves to, for example, the positive chain of n 'protein in φ χ 174 is Template synthesis In the process of copying a shape, press 3 '→ 5' to move. It plays a role in DNA replication.

DNA restriction enzyme acts on Phosphodiester bond

DNA ligase acts on phosphodiester bond

DNA Polymerase Acting on phosphodiester bond

DNA Helicase Acting on hydrogen bond

Connection means

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Three methods are known to be used in vitro Connect DNA Clip:

The first method is that DNA ligase is complementary Viscous end DNA fragments;

The second method is to use T4DNA ligase Directly Flat end DNA fragments, or Terminal deoxynucleotidyltransferase Add poly (dA) - poly (dT) tails to DNA fragments with flat ends, and then connect them with DNA ligase;

The third method is to add chemically synthesized Catcher Or joint, make them form sticky ends, and then connect them with DNA ligase. Although these three methods are different from each other, the common point is to use the function of DNA ligase to connect and block single strand DNA.

Connection of sticky end DNA fragments

The most prominent feature of DNA ligase is that it can catalyze Exogenous DNA And carrier molecules to form recombinant DNA molecule 。

Connection of flat end DNA fragments

frequently-used Flat end DNA fragment connection method, mainly including Homopolymer tailing Method, linker connection method and Joint connection method 。

Homopolymer tail adding method

The core part of this approach is to use Terminal deoxynucleotidyltransferase Special functions of transfer nucleotides. Terminal deoxynucleotidyltransferase Is from Animal tissue An abnormal DNA Polymerase , which can convert nucleotides (via Deoxynucleoside triphosphate Precursor) added to DNA molecule 3 'of single chain extension end- OH Group. from Exonuclease In the treated DNA, as well as the reaction mixture composed of dATP and terminal deoxynucleotidyltransferase, the 3 '- OH terminal of DNA molecule will appear Adenine nucleotide Consists of single strand extension of DNA. Such extended segments are called poly (dA) tails (Fig. 2-7). Conversely, if the reaction mixture is dTTP Then the 3 '- OH end of the DNA molecule will form a poly (dT) tail. Therefore, any two DNA molecules will be connected with each other as long as they obtain poly (dA) and poly (dT) tails respectively. This method of connecting DNA molecules is called Homopolymer Homopolymer tail- joining ）, referred to as homopolymer tail adding method.

Connector connection method

The so-called linker refers to a chemically synthesized segment consisting of 10-12 nucleotides with one or more restriction enzyme recognition sites Flat end Of Double chain oligonucleotides Short segments. The 5 '- end of the ligands and the 5' - end of the DNA fragments to be cloned are used Polynucleotide kinase Treatment to phosphorylate and then pass T4DNA ligase The function of makes the two connected. Then digest the ligands with appropriate restriction enzymes DNA molecule and Cloning vector Molecules, this result makes both produce complementary Viscous end 。 So we can connect the DNA fragment to be cloned with the carrier molecule according to the conventional sticky end connection method.

DNA splicing method

DNA connector is a kind of special double stranded oligonucleotide short segment with a sticky end of a restriction enzyme at one end and a flat end at the other. When its flat end is connected with the foreign DNA segment at the flat end, the latter will become a new one with sticky end DNA molecule And easy to connect and reassemble. In actual use Chemical structure Necessary modification and modification can avoid pairing connection between the sticky ends of each DNA connector molecule in the same reaction system.

DNA Polymerase

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Also called high temperature resistance DNA Polymerase 。

In 1988, Saiki went from thermophilic Aquatic fungi Thermos aquatics YT-1 strain was isolated from the United States in 1969 Huangshi National Forest Park Volcanic hot spring Is separated from.

characteristic

Good thermal stability ；

70 ℃ for 2h, residual 90% activity;

93 ℃ for 2h, residual 60% activity;

94 ℃ for 2h, 40% activity remained.

5’→3’ polymerase Activity, having preferential polymerization activity for dATP;

5’→3’ Exonuclease Activity;

No 3 '→ 5' exonuclease activity.

purpose

polymerase chain reaction (polymerase chain reaction, PCR )。

Taq enzyme The amplified PCR product always has a template independent prominence at the 3 'end Base , and this base is almost always A, because Taq enzyme has preferential polymerization activity for dATP, it can be used T vector cloning 。

shortcoming

There is no 3 '→ 5' reading correction function PCR amplification The process can cause mismatch, and the mismatch rate of 30 cycles is about 0.25%.

Action: Select High fidelity Taq enzyme , such as Pfu.

Cause: Pfu has 3 '→ 5' exonuclease activity.

Note: Pfu amplification product is Flat end 。

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