dna ligase, terminal transferase , adapter and linker

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cloning fundamentals specialy on ligation strategy

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LIGATION

SUJIT KUMAR FB-MA2-01

LIGATION OF TARGET DNA AND VECTOR

vector

insert

ligase

LIGASE JOINING OF NUCLEIC ACID EITHER DNA OR RNA- THROUGH PHOSPHODIESTER BOND. WIDESPREAD AND IDENTIFIED IN A RANGE OF ORGANISM. TWO TYPES DNA LIGASE RNA LIGASE

DNA LIGASE Dna ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination or repairing. The first DNA ligase was purified and characterized in 1967

THERE ARE LIGASES:

TWO

CLASSES

OF

DNA

THE FIRST USES NAD+ AS A COFACTOR AND ONLY FOUND IN BACTERIA. THE SECOND USES ATP AS A COFACTOR AND FOUND IN EUKARYOTES, VIRUSES AND BACTERIOPHAGES THE SMALLEST KNOWN ATP-DEPENDENT DNA LIGASE IS THE ONE FROM THE BACTERIOPHAGE T7 (MOLECULAR MASS 41 KDA).

DNA LIGASE MECHANISM THE REACTION OCCURS IN THREE STAGES IN ALL DNA LIGASES:1.FORMATION OF A COVALENT ENZYME-AMP INTERMEDIATE LINKED TO A LYSINE SIDECHAIN IN THE ENZYME. 2.TRANSFER OF THE AMP NUCLEOTIDE TO THE 5-PHOSPHATE OF THE NICKED DNA STRAND. 3.ATTACK ON THE AMP-DNA BOND BY THE 3OH OF THE NICKED DNA SEALING THE PHOSPHATE BACKBONE AND RESEALING AMP.

ACTION OF DNA LIGASE.

AN ENZYME AMP COMPLEX BINDS TO A NICK BEARING 3 OH AND 5 P GROUPS. THE AMP REACTS WITH THE PHOSPHATE GROUP. ATTACK BY THE 3 OH GROUP ON THIS MOIETY GENERATES A NEW PHOSPHODIESTER BOND, WHICH SEALS THE NICK

a) Two DNA molecules with sticky ends generated by cutting with

b) Hydrogen bonding between complementary bases causes the molecules, transiently, to stick together. DNA ligase (indicated by gray shading) catalyzes the formation of a phosphodiester bond between the 5 phosphate on one molecule and the 3 hydroxyl on the other. c) The two molecules are now covalently linked by the top strand. The nick in the bottom strand may also be sealed by DNA ligase, or may be repaired by the host bacterium.

coRI, the bases making up the EcoRI restriction site are indicated in blue.

DNA LIGASE AND GENETIC ENGINEERING In genetic engineering it is used to seal discontinuities in the sugar phosphate chains that arise when recombinant dna is made by joining dna molecules from different sources. Molecular glue - stick pieces of dna together

This function is crucial to the success of many experiments, and dna ligase is therefore a key enzyme in genetic engineering. The two most intensively studied and widely used dna ligases are E. coli dna ligase and T4 dna ligase. The enzyme used most often in experiments is t4 dna ligase, which is purified from e. Coli cells infected with bacteriophage t4.

T4 DNA LIGASE Monomeric enzyme Aminoacids- 487 Obtained from t4 bacteriophage infected e coli. Encoded by gene 30 of t4 bacteriophage. Molecular weight77000(determined by sedimentation equilibrium)

SUBSTRATECohesive termini Blunt ends DNA-RNA hybrids RNA-RNA hybrids ( 5 phosphate and 3 Hydroxyl) Rate of Blunt end ligation by T4 ligase not linearly depend upon enzyme concentration and works efficiently only in high concentration of DNA and enzyme

Condensing agent such as peg, ficoll and hexamminecobalt chloride accelerates the blunt end ligation by a factor of 1000 and permit ligation at lower enzyme, ATP and DNA concentration. Blunt end ligation is inhibited by high concentration of na(>50mm) and phosphate(>25mm)

COFACTORS Required for forming a covalent amp-enzyme intermediate. T4 DNA ligase ATP It will also utilize dAtp(at 0.5% of the rate) which acts as a competitive inhibitor with ATP

TEMPERATURE Very sensitive to the temp. Depend upon length of the joining fragment and its Tm. It has an optimum temp for ligating cohesive end of 4 degree C For sealing nick-37 degree c Blunt end ligation-25 degree c for 16 mers or longer Inactivated by heating at 65C for 10 minutes or 70C for 5 minutes

Higher level (0.2 M) of ammonium , sodium, potassium, cesium, and lithium ion inhibits completely. Unaffected by low concentration of ammonium ion Blunt end ligation is inhibited by 25 mM phosphate and 50 mM sodium ion. Polyamine such as spermine and spermidine also inhibit but can be overcome by increasing the DNA concentration.

ACTIVATORS AND INHIBITORS

Requires divalent cation for activity T4 ligase has a magnesium optimum of 10 mm whereas mn2+ is only 25% as efficient . In joining DNA:RNA hybrids Mn 2+ is twice as effective as Mg2+ Ph 40%- 6.9 65%-8.3 Optimum- 7.5 to 8.0

E COLI DNA LIGASE Encoded by lig gene of E coli The lig gene and lop11lig+ , a regulatory mutant overproducing the enzyme Monomeric, 671 amino acid Mol wt-73690 Substrate cohesive end and blunt end NO DNA-RNA or RNA-RNA hybrid

COFACTORS, INHIBITOR AND ACTIVATORS NAD as cofactor Mg2+ at an optimum concn of 1-3 mm but higher concn is inhibitory Ammonium ion at low concn stimulate it and vmax can be increased by upto 20 fold K+ and rb+ shows similar stimulation Do not required sulfhydryl reagents

Temperature-

Cohesive end- 10 to 15

COMPARISON

APPLICATION Cloning of restriction enzyme generated DNA fragments Cloning of PCR products Joining of double-stranded oligonucleotide linkers or adaptors to DNA

Site-directed mutagenesis Amplified fragment length polymorphism (AFLP) Ligase-mediated RNA detection Nick repair in duplex DNA, RNA or DNA/RNA hybrids Self-circularization of linear DNA.

LIGASE AVAILABLE IN MARKET T7 DNA Ligase Sticky-end ligation and nick sealing can be efficiently catalyzed by T7 DNA Ligase . However, unlike T4 and T3 DNA Ligases, blunt-end ligation is not efficiently catalyzed by T7 DNA Ligase, making it a good choice for applications in which blunt and sticky ends of DNA are present but only the sticky ends are to be joined.

Source-https://www.neb.com/products/dnamodifying-enzymes-and-cloningtechnologies/dna-ligases/dna-ligases

T3 DNA Ligase

Sticky ends, blunt ends, and nick sealing can all be efficiently catalyzed by T3 DNA Ligase . As with T4 DNA Ligase, blunt-end ligation is enhanced by the addition of PEG 6000 to the reaction. T3 DNA Ligase exhibits a higher tolerance (2-fold) for NaCl in the reaction compared to T4 DNA Ligase, making the enzyme a versatile choice for in vitro molecular biology protocols requiring DNA ligase activity.

ElectroLigase

ElectroLigase is specifically formulated for robust ligation of all types of DNA ends (blunt-, sticky-, T/A) and is directly compatible, without desalting or purification, with electrocompetent cells used for transformation by electroporation.

Taq DNA Ligase Taq DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini of two adjacent oligonucleotides which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them; therefore, a single-base substitution can be detected. Taq DNA Ligase is active at elevated temperatures (45C-65C)

RNA LIGASE T4 RNA Ligase catalyzes the ATPdependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'hydroxyl termini of oligonucleotides, single-stranded RNA and DNA. This enzyme is found in E coli after infection with T- seven phage. In vivo role is unclear

Monomeric enzyme Mol. Mass- 48000 (determined by sedimentation equilibrium) Nucleic acid substrate

Cofactor

Single stranded RNA It can also act on variety of single or double stranded RNA or DNA molecule It acts on very small pieces of RNA (upper limit is 40 mers) ATP Magnesium ions are required

Application RNA 3'-end labeling with cytidine 3',5'-bis [alpha-32P] phosphate Joining RNA to RNA Synthesis of oligoribonucleotides and oligodeoxyribonucleotides Specific modifications of tRNAs Oligodeoxyribonucleotide ligation to singlestranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends) Site-specific generation of composite primers for PCR

Inactivation

Inactivated by heating at 70C for 10min. Inhibitors: metal chelators, SH group-modifying reagents (8) 43.6kDa monomer

Inhibition Molecular Weight

Quality Control

The absence of ribonucleases, exodeoxyribonucleases, endodeoxyribonucleases, and phosphatases confirmed by appropriate quality tests.

Source

E.coli cells with a cloned gene 63 of bacteriophage T4

T4 RNA Ligase 2, truncated K227Q T4 RNA Ligase 2, truncated K227Q specifically ligates the preadenylated 5 end of DNA or RNA to the 3 OH end of RNA. The enzyme does not use ATP for ligation but requires pre-adenylated linkers. T4 Rnl2tr K227Q is a point mutant of T4 RNA Ligase 2, truncated . Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation (1). This mutation further reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr (2), possibly by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5-phosphates of input RNAs. The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for the cloning of microRNAs .

RNA Ligases Thermostable 5 AppDNA/RNA Ligase 5 DNA Adenylation Kit T4 RNA Ligase 1 (ssRNA Ligase) T4 RNA Ligase 2 (dsRNA Ligase) T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated K227Q T4 RNA Ligase 2, truncated KQ T4 RNA Ligase Reaction Buffer

END of PART 1

PART- 2 TERMINAL TRANSFERASE (TDT)

INTRODUCTION TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TDT) IS A TEM