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)

SUBSTRATE
Cohesive 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 TEMPLATE INDEPENDENT DNA POLYMERASE Add dNTPS to the 3-OH of oligodeoxyribonucleotides and single and double strand of DNA.

ndNTP + d(pX)m -----------d(pX)m(pN)n + nPPi (TdT and Cofactor)

TDT REQUIRES AN OLIGONUCLEOTIDE OF ATLEAST THREE NUCLEOTIDES TO SERVE AS PRIMER.

BIOLOGICAL IMPORTANCE OF TDT

Found in the nuclei of pre t and pre-b lymphocytes during immunopoiesis. It plays role during v(d)j recombination. It randomly incorporate nucleotide during v(d)j recombination and increases antigen receptor diversity and of immunoglobulin Marker enzyme of above discussed cell.

STRUCTURE
Monomeric Mol. Wt.- 60000 Da AMINO ACIDS- 508 to529(depending UPON SOURCE) A high degree of sequence homology(>80%)in tdt between different species

REACTION BUFFER
ACTIVITY IS STRONGLY INHIBITED BY THE AMMONIUM ION AS WELL AS CHLORIDE, IODIDE AND PHOSPHATE ANIONS. POTASSIUM OR SODIUM CACODYLATE(dimethyl arsenic acid) BUFFERS ARE PREFERRED- SHOWN TO BE OPTIMAL FOR POLYPURINE AND POLYPYRIMIDINE SYNTHESIS

CERTAIN DRAWBACKS WITH CACODYLATE SUCH AS TOXICITY, CONTAMINATION BY METAL etc

DIVALENT CATION
Polymerization requires presence of divalent cations. Order of efficiency for damp addition to oligonucleotide is as following Mg>Zn>Co>Mn (all are divalent cation) FOR DGTP- MAGMESIUM ION FOR PYRIMIDINE- COBALT ION

 TdT binds its substrate with high Km value of 100 micro M FOR dATP ; dGTP , 500 micro M for dTTP and dCTP ; 1 micro M for oligonucleotide primer and upto 1 mM for homopolymer primer ends.

Substrate concentration should be higher for optimal activity of TdT.. Higher concentration can be achieved by working with small volumes. The no of nucleotide added is determined by the ratio of mol dNTPs : Mol 3- OH Termini in the mixture.

Inactivation

Inactivated by heating at 70C for 10 min or by addition of EDTA. Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions

Inhibition

Quality Control

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

Source

E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase.

APPLICATION
Addition of homopolymeric tails to plasmid DNA and to cDNA Double- or single-stranded DNA 3-termini labeling with radioactively labeled or nonradioactively labeled nucleotides Addition of single nucleotides to the 3 ends of DNA for in vitro mutagenesis Production of synthetic homoand heteropolymers RACE (Rapid Amplification of cDNA Ends) Addition of vNTPs, ddNTPs and cordycepin triphosphate for chain termination in controlled manner.

Adding nucleotide tails to vector and insert DNAs using TdT

Contn.

End-labeling Using a Biotinylated Nucleotide and TdT.

Improved method for cDNA cloning. The first strand is tailed with oligo(dC) allowing the second strand to be initiated using an oligo(dG) primer

 With RNA as template TDT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using dna as a template.

(www.neb.com/products/m0315-terminal-transferase)

Terminal Transferase

Terminal transferase (TdT) is a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. Protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as a substrate for TdT. The 58.3 kDa enzyme does not have 5' or 3' exonuclease activity. The addition of Co2+ in the reacton makes tailing more efficient. Highlights
Isolated from a recombinant source Labeling of the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP) Supplied with 10X Reaction Buffer and 2.5mM CoCl2

 Product Source An E. coli strain that carries the cloned Terminal

Applications

Transferase gene from calf thymus. Reagents Supplied The following reagents are supplied with this product: CoCl2 ............10X Terminal Transferase Reaction Buffer................10X Addition of homopolymer tails to the 3' ends of DNA Labeling the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP) TUNEL assay (in situ localization of apoptosis) TdT dependent PCR

END OF PART- 2

PART 3 ADAPTER AND LINKER

INTRODUCTION
Ligation efficiency depends on the DNA ends in the reaction
Complementary sticky ends Ligation is efficient annealing of complementary overhangs brings 5P and 3OH into close proximity Blunt ends Ligation is inefficient need high concentrations of ligase and DNA molecular crowding reagents (like PEG 8000) improve intermolecular ligation, then dilute to promote intramolecular ligation

Cloning strategy

BLUNT END LIGATION

Under these circumstances one of following three methods can be used to put the correct sticky ends onto the DNA fragments-

Terminal transferase to add polynucleotide tails to foreign DNA and vector DNA Cloning foreign DNA by adding linkers Cloning foreign DNA by adding adaptors

LINKER
Linkers are the chemically synthesized double stranded DNA oligonucleotides containing on it one or more restriction sites for cleavage by restriction enzymes, e.g. Eco RI, Hind III, Bam HI, etc. Linkers are ligated to blunt end DNA by using T4 DNA ligase. Both the vector and DNA are treated with restriction enzyme to develop sticky ends. The staggered cuts i.e. sticky ends are then ligated with T4 DNA ligase with very high efficiency to the termini of the vector and recombinant plasmid DNA molecules are produced.

EcoRI linker:

:d(GGAATTCC) 8 :d(CGGAATTCCG) 10 :d(CCGGAATTCCGG) 12

Add 2,4,6 "extra" base pairs in addition to the restriction site. This changes reading frame. ATG ATG ATG ATG if introduced between middle pair: (Met-Met-MetMet)

Why different size linkers?

=

=ATG ATG CGG AAT TCC GAT GAT G 10-mer (Met-Met-Arg-AsnSer-Asp-Asp) =ATG ATG CCG GAA TTC CGG ATG ATG 12-mer (Met-Met-ProGlu-Phe-Arg-Met-Met).

end)

ATG ATG GGA ATT CCA TGA TG 8-mer (Met-Met-Gly-Asn-Pro-

Any linker divisible by 3 should not change reading frame

LIMITATIONS OF LINKER
It may be the case that the restriction enzyme used to generate the cohesive ends in the linker will also cut the foreign DNA at internal sites.
CHOOSE ANOTHER RESTRICTION ENZYME

But there may not be a suitable choice if the foreign DNA is large and has sites for several restriction enzymes.

Methylate internal restriction sites with the Appropriate modification methylase for example EcoRI methylase.

ADAPTER
Adapters are also the chemically synthesized partialy double stranded DNA oligonucleotides, with a blunt end at one side, and a cohesive end at the other side which contains a recognition site for a restriction enzyme.
Using T4 DNA ligase, adapters can be added to a blunt-ended DNA fragment

 But unlike linkers, an adaptor is synthesized so that it already has one sticky end.

Adaptors: Duplexes that are partially ds and partially ss.

BamHI adapter
5p-GATCCCGG-OH3 GGCC-p5

The idea is of course to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to produce a new molecule with sticky ends.

 This may appear to be a simple method but in practice a new problem arises; The sticky ends of individual adaptor molecules could base pair with each other to form dimers, so that the new DNA molecule is still blunt ended. The sticky ends could be recreated by digestion with a restriction endonuclease, but that would defeat the purpose of using adaptors in the first place. The answer to the problem lies in the precise chemical structure of the ends of the adaptor molecule

Adaptors and the potential problem with their use. (a) A typical adaptor. (b) Two adaptors could ligate to one another to produce a molecule similar to a linker (c) after ligation of adaptors a blunt-ended molecule is still bluntended and the restriction step is still needed

 Normally the two ends of a polynucleotide strand are chemically distinct, a fact that is clear from a careful examination of the polymeric structure of DNA. One end, referred to as the 5' terminus, carries a phosphate group (5'-P); the other, the 3 terminus, has a hydroxyl group (3'-OH). In the double helix the two strands are antiparallel, so each end of a double-stranded molecule consists of one 5'-P terminus and one 3'-OH terminus.

 Ligation normally takes place between the 5'-P and 3'-OH ends. Adaptor molecules are synthesized so that the blunt end is the same as natural DNA, but the sticky end is different. The 3'- OH terminus of the sticky end is the same as usual, but the 5'P terminus is modified; it lacks the phosphate group (removed by Alkaline Phosphates treatment), and is in fact a 5'-OH terminus. DNA ligase is unable to form a phosphodiester bridge between 5'-OH and 3'-OH ends.

 The result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized by ligation. Adaptors can therefore be ligated to a DNA molecule but not to themselves. After the adaptors have been attached, the abnormal 5'-OH terminus is converted to the natural 5'-P form by treatment with the enzyme polynucleotide kinase, producing a sticky-ended fragment that can be inserted into an appropriate vector.

REFERENCE
Primrose S.B. & Twyman R.M. 2006. Principles of gene manipulation and genomics. 7th ed. Blackwell publishing, Malden. Brown, T.A. (Terence A.).2010.Gene cloning and DNA analysis : an introduction / T.A. Brown.6th ed. John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex UK Nicholl,Desmond S. T. 2008. An Introduction to Genetic Engineering,Third Edition, CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, So Paulo https://www.neb.com