NA LIGAS ANALYSIS - Lecture Notes - TIU

Biotechnology-Theory Lec. 7 3rd Student-Medical Analysis 2021

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DNA LIGASE ANALYSIS

Objectives: Students should able to know and explain;

What are DNA Ligases?

How do DNA Ligases work?

What is the principle mechanism of Ligation Reaction?

How many methods are used for joining or inserting of DNA to vector?

What are the applications of DNA Ligase?

Aim: Join of foreign DNA fragment to a cloning vector to create a recombinant DNA.

DNA Ligase:

Since they are involved in such important processes as DNA replication, DNA

repair, and DNA recombination. DNA ligases can be found in all living cells

(Both Prokaryotes and Eukaryotes). Two prokaryotic DNA ligases have become

indispensable tools in the fields of in vitro DNA recombination and DNA

synthesis.

These two enzymes which are extensively used for covalently joining

restriction fragments include: The ligase from E. coli and that encoded by T4

phage. The main source of DNA ligase is T4 phage, hence, the enzyme is known

as T4 DNA ligase.

DNA ligases seal and join together the ends of the foreign DNA and vector

already digested with the same restriction enzyme. Both ligase enzymes catalyze

the synthesis of a covalent phosphodiester bond between the 3’-hydroxyl group

and the 5’-phosphoryl group on various substrates such as DNA nicks, DNA

fragments with various lengths cohesive ends, DNA fragments with blunt ends

and some DNA/RNA hybrids (Figure 1). RNA ligases also exist, but are rarely

used in biotechnology.

The substrates may be DNA or RNA, and the cofactors that generate high-

energy intermediates in the reaction may be ATP (adenosine triphosphate) or

NAD' (nicotinamide adenine dinucleotide), depending on the type of ligase, that

is ATP-dependent DNA Ligases and NAD+-dependent DNA Ligases (Figure 2).

Subsequent studies have revealed the existence of both NAD + - and ATP

dependent DNA ligases in prokaryotes. In contrast, eukaryotic and viral DNA

ligases are almost exclusively ATP-dependent (for example, mammalian DNA

ligases).


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Difference between E. coli DNA Ligase and T4 DNA Ligase


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Summarizing of DNA Ligase Analysis:

ο The discovery of DNA ligases in 1967 by the Gellert, Lehman, Richardson,

and Hurwitz laboratories was a watershed event in molecular biology. They

identified DNA ligase activity in extracts from uninfected E. coli cells and also

from E. coli cells infected with bacteriophage T4.

ο DNA ligases are essential guardians of genomic integrity, and ligase

dysfunction underlies human genetic disease syndromes.

ο They are essential for DNA replication and repair in all organisms.

ο Ligases were critical reagents in the development of molecular cloning and

many subsequent ramifications of DNA biotechnology, including molecular

diagnostics (e.g. Ligase Chain Reaction (LCR) and Ligase Detection Reaction

(LDR)), Solid sequencing methods, DNA labeling, mutagenesis, and other in

vitro DNA manipulations.

ο Ligases are elegant and versatile enzymes and are enjoying a research

renaissance in light of discoveries that most organisms have multiple ligases

that either function in DNA replication (by joining Okazaki fragments) or are

dedicated to particular DNA repair pathways, such as nucleotide excision

repair, base excision repair, single-strand break repair, or the repair of double

strand breaks via non-homologous end joining.

ο Once the DNA to be cloned exists as a defined fragment (called the target or

insert), it can be joined to the vector by the process called ligation.

ο The desired product in a ligation reaction is a functional hybrid molecule that

consists exclusively of the vector plus the insert.

ο Dugaiczyk et al. (1975) described the events occurring during ligation of

EcoR I fragments, observations that can be applied to any ligation reaction.


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ο Two vector types are used in recombinant DNA research, circular molecules

(vectors) such as plasmids and cosmids and linear cloning vectors such as

those derived from the bacteriophage lambda.

ο In either case, for joining to a target DNA fragment, a vector is first cut with

a restriction enzyme that produces compatible ends with those of the target.

Circular vectors, therefore, are converted to a linear form prior to ligation to

target.

ο The insert fragment is then ligated to the prepared vector to create a

recombinant molecule capable of replication once introduced into a host cell

(Competent Cell).

o THE RANGE OF DNA MANIPULATIVE ENZYMES

DNA manipulative enzymes can be grouped into four broad classes, depending

on the type of reaction that they catalyse:

Nucleases: are enzymes that cut, shorten, or degrade nucleic acid molecules.

LIGASES: JOIN NUCLEIC ACID MOLECULES TOGETHER.

Polymerases: make copies of molecules.

Modifying enzymes: remove or add chemical groups.

Within the cell, the function of DNA ligase is to repair single-stranded breaks

(‘discontinuities’) that arise in double-stranded DNA molecules during, for

example, DNA replication. DNA ligases from most organisms can also join

together two individual fragments of double-stranded DNA (Figure 3). These

enzymes have the role of in construction of recombinant DNA molecules.

Figure 3

The two reactions catalysed by DNA

ligase. (a) Repair of a discontinuity – a

missing phosphodiester bond in one

strand of a double-stranded molecule.

(b) Joining two molecules together.


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After cutting the source DNA to generate fragment of interest, the next task of

cloning is generation of recombinant DNA molecule by joining DNA fragment

of interest to appropriate vector and it can be transferred to suitable host

(Competent Cell). However, the plasmids, phages and cosmids are most

commonly used depending on their suitability for different kinds of work.

Following methods are most commonly used for joining or inserting of DNA

fragment to vector:

1. Sticky or cohesive end (termini) ligation.

2. Blunt End Ligation (No linker used).

3. Homopolymer tailing.

4. Use of linker molecules (Single or double).

5. Use of adaptor molecules.

ο LIGATION: JOINING DNA MOLECULES TOGETHER:

The final step in the construction of a recombinant DNA molecule is a joining

together of the vector molecule and the DNA to be cloned (Figure 4). This

process is referred to as ligation, and the enzyme that catalyzes the reaction is

called DNA ligase.

Cloning vector: A vector is an agent that can carry DNA fragment into a host

cell. If the DNA fragment have the ability to replicate in an appropriate host cell,

it is called a cloning vector. If it is used for expressing certain gene in the DNA

fragment, it is called an expression vector.

FIGURE 4. Ligation: the final step in construction

of a recombinant DNA molecule.


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THE MODE OF ACTION OF DNA LIGASE: All living cells produce DNA ligases, but the enzyme used in genetic

engineering is usually purified from E. coli bacteria that have been infected

with T4 phage. Within the cell, the enzyme carries out the very important

function of repairing any discontinuities that may arise in one of the strands

of a double-stranded molecule (see Figure 3).

A discontinuity is quite simply a position where a phosphodiester bond

between adjacent nucleotides is missing (contrast this with a nick, where one

or more nucleotides are absent).

Although discontinuities may arise by a chance breakage of the cell’s DNA

molecules, they are also a natural result of processes such as DNA replication

and recombination. Ligases therefore play several vital roles in the cell.

In the test tube, purified DNA ligases not only repair single-strand

discontinuities but also join together individual DNA molecules, or the two

ends of the same molecule and this is called BLUNT END LIGATION

METHOD (NO LINKER USED).

The chemical reaction involved in ligating two molecules is exactly the same

as discontinuity repair, except that two phosphodiester bonds must be made,

one for each strand (see Figure 5).

STICKY ENDS INCREASE THE EFFICIENCY OF LIGATION

(STICKY OR COHESIVE END (TERMINI) LIGATION) METHOD:

The ligation reaction in Figure 5 shows two blunt-ended fragments being

joined together. Although this reaction can be carried out in the test tube, it is

not very efficient because the ligase is unable to ‘catch hold’ of the molecule

to be ligated and so must wait for chance associations to bring the ends

together.

If possible, blunt end ligation should be performed at high DNA

concentrations, to increase the chances of the ends of the molecules coming

together in the correct fashion.

Figure 5

Ligation of blunt-ended DNA molecules by DNA ligase.


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In contrast, the ligation of complementary sticky ends is much more

efficient. This is because compatible sticky ends can base pair with one another

by hydrogen bonding (see Figure 6), forming a relatively stable structure for

the enzyme to work on.

If the phosphodiester bonds are not synthesized fairly quickly the sticky ends

will fall apart again. Nonetheless, these transient, base-paired structures

increase the efficiency of ligation by increasing the period of time that the ends

are in contact with one another.

PUTTING STICKY ENDS ON TO A BLUNT-ENDED MOLECULE:

For the reasons detailed in previous section above, compatible sticky ends

are desirable on the DNA molecules to be ligated together in a gene cloning

experiment. Often, these sticky ends can be provided by digesting both the

vector and the DNA to be cloned with the same restriction endonuclease, or

with different enzymes that produce the same sticky end, though it is not

always possible to do this.

A common situation is where the vector molecule has sticky ends but the

DNA fragments to be cloned are blunt-ended. Under these circumstances,

one of three methods can be used to put the correct sticky ends onto the DNA

fragments.

Figure 6

Ligation of sticky-ended molecules.


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1. Linkers:

ο Linkers are short pieces of double-stranded DNA of known nucleotide

sequence that are synthesized in the test tube. A typical linker, as shown in

Figure 7a, is blunt-ended but contains a restriction site (BamHI in the

example shown). DNA ligase can attach linkers to the ends of larger blunt-

ended DNA molecules.

ο Although a blunt end ligation, this particular reaction can be performed very

efficiently because synthetic oligonucleotides, such as linkers, can be made

in very large amounts and added into the ligation mixture at a high

concentration.

ο More than one linker will attach to each end of the DNA molecule, producing

the chain structure shown in Figure 7b.

ο Digestion with BamHI cleaves the chains at the recognition sequences,

producing a large number of cleaved linkers and the original DNA fragment,

now carrying BamHI sticky ends. This modified fragment is ready for ligation

into a cloning vector restricted with BamHI restriction enzymes.

Figure 7 Linkers and their use. (a) The structure of a typical linker. (b) The attachment of linkers to

a blunt-ended molecule.


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2. Adaptors:

ο The use of linkers has one potential drawback. If the blunt-ended molecule

shown in Figure 7b were to contain one or more BamHI recognition sequences,

then the restriction step needed to cleave the linkers and produce the sticky

ends would also cleave the blunt-ended molecule (Figure 8).

ο The resulting fragments would have the correct sticky ends, but that would be

no consolation if the gene contained in the blunt-ended fragment had now been

broken into pieces.

ο The second method (An adaptor) of attaching sticky ends to a blunt-ended

molecule is designed to avoid this problem.

ο An adaptor, like a linker, is a short synthetic oligonucleotide but is synthesized

so that it already has one sticky end (Figure 9a). 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.

ο The sticky ends of individual adaptor molecules could base pair with each

other to form dimers (Figure 9b) so that the new DNA molecule is still blunt-

ended (Figure 9c). Although the sticky ends could be recreated by digestion

with a restriction endonuclease, that would defeat the purpose of using

adaptors in the first place.

Figure 8

A possible problem with the use of

linkers. Compare this situation with

the desired result of BamHI

restriction, as shown in Figure 7b.

Figure 9

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, so that (c) after

ligation of adaptors a blunt-ended molecule is

still blunt-ended and the restriction step is still

needed.


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ο The answer to this problem lies in the precise chemical structure of the ends of

the adaptor molecule. 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 (Figure 10a). One end, referred to as the 5′

terminus, carries a phosphate group (5′-P), while the other, the 3′ terminus, has

a hydroxyl group (3′-OH). In the double helix the two strands are antiparallel

(Figure 10b), so each end of a double-stranded molecule consists of one 5′-P

terminus and one 3′-OH terminus. Ligation then takes place between the 5′-P

and 3′-OH ends (Figure 10c).

ο 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, and is in fact a 5′-OH terminus (Figure 11a).

ο 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 (Figure 11b).

Figure 10

The distinction between the 5′ and 3′ termini of a polynucleotide.


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ο Adaptors can therefore be ligated to a blunt-ended 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.

3. Homopolymer tailing:

ο The technique of homopolymer tailing offers a radically different approach to

the production of sticky ends on a blunt-ended DNA molecule.

ο A homopolymer is simply a polymer in which all the subunits are the same.

A DNA strand made up entirely of, say, deoxyguanosine is an example of a

homopolymer, and is referred to as polydeoxyguanosine or poly(dG).

ο Tailing involves using the enzyme terminal deoxynucleotidyl transferase to

add a series of nucleotides onto the 3′-OH termini of a double-stranded DNA

molecule (Figure 12a and b).

ο In practice, the poly(dG) and poly(dC) tails are not usually exactly the same

length, and the base-paired recombinant molecules that result have nicks as

well as discontinuities (Figure 12c).

ο Repair is therefore a two-step process, using Klenow polymerase to fill in the

nicks, followed by DNA ligase to synthesize the final phosphodiester bonds.

Figure 11

The use of adaptors. (a) The actual structure of an

adaptor, showing the modified 5′-OH terminus. (b)

Conversion of blunt ends to sticky ends through the

attachment of adaptors.


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Figure 12

Homopolymer tailing. (a) Synthesis of a

homopolymer tail. (b) Construction of a

recombinant DNA molecule from a tailed

vector plus tailed insert DNA. (c) Repair of the

recombinant DNA molecule.


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Your Homework and Assessment:

Q1) Explain the following terms:

1. DNA linkers. 2. DNA adapter. 3. Recombinant DNA.

Q2) What are the main differences between the DNA ligase and T4 DNA Ligase?

Q3) How many types of DNA ligases?

Q4) What is the meaning THE LIGASE CHAIN REACTION (LCR)?

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NA LIGAS ANALYSIS - Lecture Notes - TIU