DNA ligase is an essential enzyme that joins together DNA fragments. The first DNA ligase was purified in 1967. It plays a key role in processes like DNA replication, repair, and recombinant DNA techniques. There are different types of ligases from sources like E. coli and T4 bacteriophage. Ligases can join DNA fragments with complementary sticky ends or blunt ends, and are used to integrate DNA into vectors for cloning. T4 ligase is most commonly used as it acts on a broad range of substrates.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.
Transferring the genetic information to the descendant generation with a high fidelity
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
THE MECHANISM OF DNA POLYMERASE & CHEMICAL NATURTE TOPIC OF MOLECULAR BIOLOG...Lucky234529
WELCOME TO LOVYANSH LIFE SCIENCE
DETAIL EXPLANATION OF DNA POLYMERASE IN WHICH MY YOU TUBE LECTURE PLEASE CLICK A LINK & SEE YOU TUBE VIDEO .
https://youtu.be/kKFOHcq6nTM
Recombinant dna technology and DNA sequencinganiqaatta1
title: recombinant DNA technology and DNA sequencing
this lect will cover the pcr, isolation of DNA, detection of DNA and DNA manipulation joining DNA together. this is very important and it is required in research of every field especially medical related field.
Transgenic animal production and its applicationkishoreGupta17
A genetically modified animal with the heterologous gene of interest being inserted for the purpose of biopharming or make a diseased model to study the consequences of disease and its probable therapy
for cloning and expression of exogenous gene or gene throthrough vector it must be introduced into the host cell through transformation , ,transduction, electroporation gene gun etc.
A genetically engineered DNA molecule from bacteria , phage or yeast to carry foreign DNA for the purpose of cloning and expression of the inserted DNA of interest in RDT
A cytological technique to detect the nature of adjacent chromosomal regions by using different staining technique assisted with some pre treatment of metaphase chromosomes prepared on the slides
How to Create Map Views in the Odoo 17 ERPCeline George
The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
The Art Pastor's Guide to Sabbath | Steve ThomasonSteve Thomason
What is the purpose of the Sabbath Law in the Torah. It is interesting to compare how the context of the law shifts from Exodus to Deuteronomy. Who gets to rest, and why?
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
2. Introduction
• First DNA ligase was purified and characterized in 1967 by the Gellert,
Lehman, Richardson, and Hurwitz laboratories.
• Although cutting DNA precisely by RE is useful for DNA analysis but its full
potential is only rely when the fragments produced by RE are joined
together to form recombinant DNA..
• This joining is achieved by DNA ligase.
• Ligase is an enzyme to join any two DNA segment together and process is
called ligation despite of their different origin
For cloning and expression the genomic integrity of vector is
essential,
3. Continued…..
• DNA ligases play an essential role in maintaining
genomic integrity by joining breaks in the
phosphodiester backbone of DNA that occur during
• replication and recombination, and
• Repair of damaged DNA.
• In Biotechnology used to Integrate the DNA fragments
in to the vector
•
4.
5. Source of ligase .
• Though ligase is of universal occurrence in
living organism and viruses but for use in RDT
it is isolated from two sources
• E.coli. 75 Kda.
• T4 bacteriophage 68 KDa - first ATP-
dependent ligase enzyme to be discovered
and is widely used in molecular biology,
• The enzyme isolated from bacteria E.coli is a
polypeptide chain with mol.wt of
6. Types of ligase
• E.coli DNA ligase -Monomeric , MW=74 kDa
• T4 Ligase
• Ampligase
• Genetically engineered ligase
• The RNA ligase for RNA fragments ligation .catalyzes
the formation of 3′ → 5′ phophodiester bondsetween
3′-OH and 5′-P groups of RNA molecules.
• Repairing of Anticodon loop
•
7. Substrate of ligase
various substrates such as DNA nicks, DNA
fragments with various lengths cohesive ends,
DNA fragments with blunt ends and some
DNA/RNA hybrids.
8. Ligase in Biotech
DNA Ligase
• + In all except Viruses but only
E coli DNA ligase is used in
biotech .
• Use NAD as cofactor
• Can not ligate blunt end DNA
fragments
• cannot join RNA to DNA
efficiently
• Narrowness activity (specifity
for chohesive ends joining
T4 Ligase
• Bacteriophage and most
commonly used
• ATP as cofactor
• ligate either cohesive or blunt
ends of DNA fragments , as
well as RNA and RNA-DNA
hybrids, but not single-
stranded nucleic acids
• Ligate blunt ended DNA with
much greater efficiency than E
coli DNA ligase
• Can not join ss DNA
• Also join ss nick in ds DNA and
RNA-DNA Hybrud
• Broad activity
9. Genetically engineered T4 Ligase
• Some engineering has been done to improve
the in vitro activity of T4 DNA ligase; one
successful approach, for example, tested T4
DNA ligase fused to several alternative DNA
binding proteins and found that the constructs
with either p50 or NF-KB as fusion partners
were over 160% more active in blunt-end
ligations for cloning purposes than wild type
T4 DNA ligase
10. Ligation efficiency
Depends upon types of end sticky/ blunt
Sticky –efficient
Blunt –less efficient and need more concentration of ligase
11. Activity and Mechanism of ligase action
• If two different DNA are treated with same restriction enzyme to give fragments
with complementary cohesive ends /sticky ends and mixed then two DNA will join
by forming phosphester bond between terminal nucleotides (5’phophoryl at the
end of one strand i.e donor )and 3’OH at other end i.e acceptor but it has nick
after
ATP is required which proceed in three steps
• 1.adenylation –addition of AMP in the active centre of ENz. And PP is released
2.,transfer of the AMP to the 5”P so called donor and formation of pyrophosphate
bond
• 3. Formation of phosphodiester bonds )
• T4 ligase catalyzes end to end joining.
• This could occur inter/intra molecular but inter molecular joining is correct.
• Ligation requires ATP and is carried at 4 deg celcius to lower the kinetic energy.
• This reduce the separation of paired sticky end which is later stabilized by ligation.
However, long reaction times are needed to compensate the low activity of DNA
ligase in cold.
13. Weiss unit -
The amount of ligase that catalyzes the
exchange of 1 nmole of 32P from
inorganic pyrophosphate to ATP in 20
minutes at 37°C.
This is the one most commonly used.
14. enhancer for DNA ligase
• The activity of E. coli DNA ligase can be enhanced
by DNA POL at the right concentrations.
Enhancement only works when the
concentrations of the DNA polymerase 1 are
much lower than the DNA fragments to be
ligated. When the concentrations of Pol I DNA
polymerases are higher, it has an adverse effect
on E. coli DNA ligase
• The T4 enzyme conc is kept high and PEG is
added to reaction mixture for stimulation.
15. Blunt end ligation
• `
• Blund end fragment has no overhang which can
hold the two fragment temporarily together.
• So ligase and DNA concentration are kept high .
• It is therefore used to ligate the two DNA
fragments obtained by two different restriction
enzyme with non compatible sticky ends DNA.
The non complementary DNA are removed prior
to ligation using enzyme SI nuclease which digest
SS DNA
16. For this
• Blunt ended fragments are linked with the linker and adapter
before joining
• Linkers & Adaptors
• Recombinant DNA and gene cloning use RE to cut DNA at specific
site .
• If suitable sites are not available for manipulation then these
cleavage sites can be added as linker or adaptor
• Linkers are synthetic SS DNA of 8-14 bases with Res. Sites for 3-8
RE which will self reassociate to form DS DNA
• For eg 5’ CCGAATTCGG 3’ CCGAATTCGG
• GGCTTAAGCC
• It contains ECOR I site . This sequence can be ligated to blunt end
and then digested with Rest enz. Cohesive end is produced.
17.
18. Application
• DNA ligase is important enzymatic tools without which RDT can not be achieved.
• The important function are:-
• Involves in ligation or joining of two DNA segments.
• Like –
• Two okazaki fragments in replication The fragments are formed of lagging strand and after removal of
primer the DNA segments are joined together two give continuous strand of DNA against 5’-3’ template
strand.
• It is used in Ligation of DNA in Vector for cloning & expression.
• Used in Ligation of linker and adaptor at the blunt ends of DNA fragments
• Used in Sealing of nick in DNA fragments
• 2. It I also used in ELISA to form Enzyme substare conjugate . P-nitrophenyl phosphate is a substrate
for AP but cannot form conjugate to give coloured product . The Ap remove P and forms a conjugate
• Note .
• Self ligation is prevented by use of Alkaline phosphatase. De phophorylated Vector DNA cannot be
recircularised in the cloning procedure and can be remain linear for ligating with the other DNA.
• Ligase requires 3’OH and 5’PO4 for ligation