LIGATION

SUJIT KUMARFB-MA2-01

LIGATION OF TARGET DNA AND VECTOR

insertligase

vector

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 TWO CLASSES OF DNA LIGASES: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 SIDE-CHAIN 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 3’-OH 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 WITHTHE 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 coRI, the bases making up the EcoRI restriction site are indicated in blue.b) Hydrogen bonding between complementary bases causes the molecules,transiently, to stick together. DNA ligase (indicated by gray shading) catalyzes theformation of a phosphodiester bond between the 5′ phosphate on one moleculeand the 3′ hydroxyl on the other. c) The two molecules are now covalentlylinked by the top strand. The nick in the bottom strand may also be sealed byDNA ligase, or may be repaired by the host bacterium.

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 65°C for

10 minutes or 70°C for 5 minutes

ACTIVATORS AND INHIBITORS• 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.

• 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

• Cloning of restriction enzyme generated DNA fragments

• Cloning of PCR products• Joining of double-stranded

oligonucleotide linkers or adaptors to DNA

APPLICATION

• 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.

• 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/dna-modifying-enzymes-and-cloning-technologies/dna-ligases/dna-ligases

LIGASE AVAILABLE IN MARKET

• 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 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 (45°C-65°C) 

Taq DNA Ligase

• T4 RNA Ligase catalyzes the ATP-dependent 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

RNA LIGASE

• Monomeric enzyme• Mol. Mass- 48000 (determined by sedimentation

equilibrium)• Nucleic acid substrate

– 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)• Cofactor

– ATP– Magnesium ions are required

• 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 single-

stranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends)

• Site-specific generation of composite primers for PCR

Application

Inactivation Inactivated by heating at 70°C for 10min.

Inhibition Inhibitors: metal chelators, SH group-modifying reagents (8)

Molecular Weight 43.6kDa monomer

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 specifically ligates the pre-adenylated 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 .

T4 RNA Ligase 2, truncated K227Q

• 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

Welcome again

PART- 2 TERMINAL

TRANSFERASE (TDT)

• 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.

INTRODUCTION

• 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.

BIOLOGICAL IMPORTANCE OF TDT

• Monomeric• Mol. Wt.- 60000 Da• AMINO ACIDS- 508 to529(depending UPON

SOURCE)• A high degree of sequence

homology(>80%)in tdt between different species

STRUCTURE

• 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

REACTION BUFFER

• 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 IONFOR PYRIMIDINE- COBALT ION

DIVALENT CATION

• 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 70°C for 10 min or by addition of EDTA.

Inhibition Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions

Quality Control

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

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

• Addition of homopolymeric tails to plasmid DNA and to cDNA

• Double- or single-stranded DNA 3´-termini labeling with radioactively labeled or non-radioactively labeled nucleotides

• Addition of single nucleotides to the 3´ ends of DNA for in vitro mutagenesis

• Production of synthetic homo- and heteropolymers• RACE (Rapid Amplification of cDNA Ends)• Addition of vNTPs, ddNTPs and cordycepin

triphosphate for chain termination in controlled manner.

APPLICATION

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.

• 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

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

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

gene from calf thymus.Reagents Supplied– The following reagents are supplied with this product:– CoCl2 ............10X– Terminal Transferase Reaction Buffer................10X

• Applications– 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 – 3ADAPTER AND LINKER

• Ligation efficiency depends on the DNA ends in the reaction

• Complementary “sticky” ends• Ligation is efficient• annealing of complementary overhangs brings 5’P and 3’OH 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

INTRODUCTION

Cloning strategy

• 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

BLUNT END LIGATION

• 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.

LINKER

EcoRI linker:• :d(GGAATTCC) 8• :d(CGGAATTCCG) 10• :d(CCGGAATTCCGG) 12

Why different size linkers? – 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-Met-Met)

= ATG ATG GGA ATT CCA TGA TG 8‑mer (Met-Met-Gly-Asn-Pro-end) =ATG ATG CGG AAT TCC GAT GAT G 10‑mer (Met-Met-Arg-Asn-Ser-Asp-

Asp) =ATG ATG CCG GAA TTC CGG ATG ATG 12‑mer (Met-Met-Pro-Glu-Phe-

Arg-Met-Met).

• Any linker divisible by 3 should not change reading frame

• 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

LIMITATIONS OF LINKER

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.

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

ADAPTER

• 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

• 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.

5‘p-GATCCCGG-OH3’ GGCC-p5’

• 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 couldligate to one another to produce a molecule similarto a linker (c) after ligation of adaptors ablunt-ended molecule is still blunt-ended and therestriction 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.

• 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, São Paulo

• https://www.neb.com

REFERENCE

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