This document discusses enzymes used in genetic engineering, specifically nucleases. It describes how nucleases can cleave DNA in specific ways, enabling manipulation of DNA in vitro. It classifies nucleases as exonucleases, which degrade DNA from the ends, or endonucleases, which cleave internally. Restriction enzymes are a type of endonuclease that recognize specific DNA sequences and are critical tools in genetic engineering. The document outlines the different types of restriction enzymes and how they cleave DNA to produce blunt or sticky ends, enabling cutting and joining of DNA fragments.
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
BRIEFLY EXPLAINED PPT ABOUT RESTRICTION ENZYMES, THEIR WORKING SITES, TYPES, ARTIFICIALLY GENERATED RESTRICTION ENZYMES, THEIR MECHANISM OF ACTION, TYPES OF CUTS THEY MAKE, THEIR NOMENCLATURE ETC.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
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
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
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.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
BRIEFLY EXPLAINED PPT ABOUT RESTRICTION ENZYMES, THEIR WORKING SITES, TYPES, ARTIFICIALLY GENERATED RESTRICTION ENZYMES, THEIR MECHANISM OF ACTION, TYPES OF CUTS THEY MAKE, THEIR NOMENCLATURE ETC.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
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
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
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.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
Also referred to as Restriction Endonucleases
Molecular scissors that cut double stranded DNA molecules at specific points.
Found naturally in a wide variety of prokaryotes
An important tool for manipulating DNA.
Enters and recognizes a certain sequence on a double helix strand of DNA, usually 4-6 base-pairs long, and cuts it.
Precise by cutting both strands in same location though strands move in reverse directions; REs are able to depict the precise spot to cut
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
3. Nucleases: Exonucleases and Endonucleases
• The ability to manipulate DNA in vitro depends entirely on the availability of purified enzymes
that can cleave, modify and join the DNA molecule in specific ways.
• At present, no chemical method can achieve the ability to manipulate the DNA in vitro in a
predictable way.
• Only enzymes are able to carry out the function of manipulating the DNA. Each enzyme has a
vital role to play in the process of genetic engineering.
• The various enzymes used in genetic engineering are as follows:
• Nucleases
• Restriction enzymes
• DNA modifying enzymes
• DNA Ligase
• Polymerase
• Topoisomerase
4. NUCLEASES
• ‘Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA
strand.
• In addition to their important biological role, nucleases have emerged as useful tools in laboratory studies, and have led
to the development of such fields as recombinant DNA technology, molecular cloning, and genomics’.
• “Processes under control of nucleases are for example
• protective mechanisms against "foreign" (invading) DNA,
• degradation of host cell DNA after virus infections,
• DNA repair,
• DNA recombination,
• DNA synthesis
• DNA packaging in chromosomes and viral compartments,
• maturation of RNAs or RNA splicing.
5. NUCLEASES - CLASSIFICATION
• They are classified by their specificity of their requirement for either a free end (exo)
to start working or they start from anywhere within a molecule (endo) even when no
free ends are available as for example in a covalently closed circle
• EXONUCLEASES:
• Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA
molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I,
exonuclease II etc.
• The main distinction between different exonucleases lies in the number of strands
that are degraded when a double-stranded molecule is attacked.
• For example B al31 degrades both strand
• E. coli exonuclease III degrades only one strand and only from the 3′ terminus.
6. Figure 4.1 The reactions catalysed by the two different kinds of
nuclease. (a) An exonuclease, which removes nucleotides from
the end of a DNA molecule. (b) An endonuclease, which breaks
internal phosphodiester bonds.
7.
8.
9. ENDONUCLEASES
• Endonucleases can recognize specific base sequence (restriction site)
within DNA or RNA molecule and cleave internal phosphodiester bonds
within a DNA molecule.
• Example: E coRI, H ind III, B amHI etc.
10. RESTRICTION ENZYMES
• DNases which act on specific positions or sequences on the DNA are called as
restriction endonucleases.
• The sequences which are recognized by the restriction endonucleases or restriction
enzymes (RE) are called as recognition sequences or restriction sites. These sequences
are palindromic sequences.
• The discovery of these enzymes, led to Nobel Prizes for W. Arber, H. Smith, and D.
Nathans in 1978
• Restriction endonucleases are synthesized by many, perhaps all, species of bacteria:
over 2500 different ones have been isolated and more than 300 are available for use
in the laboratory.
11.
12. MODE OF ACTION
• The restriction enzyme binds to the recognition site and checks for the
methylation (presence of methyl group on the DNA at a specific
nucleotide). If there is methylation in the recognition sequence, then, it
just falls off the DNA and does not cut.
• If only one strand in the DNA molecule is methylated in the recognition
sequence and the other strand is not methylated, then RE (only type I and
type III) will methylate the other strand at the required position.
• The methyl group is taken by the RE from S-adenosyl methionine by using
modification site present in the restriction enzymes.
13. MODE OF ACTION
• However, type II restriction enzymes take the help of another enzyme called
methylase, and methylate the DNA.
• Then RE clears the DNA. If there is no methylation on both the strands of DNA,
then RE cleaves the DNA.
• It is only by this methylation mechanism that, RE, although present in bacteria,
does not cleave the bacterial DNA but cleaves the foreign DNA.
•
• But there are some restriction enzymes which function exactly in reverse mode.
• They cut the DNA if it is a methylate.
14. NOMENCLATURE OF RESTRICTION ENZYMES
• As a large number of restriction enzymes have been discovered, a
uniform nomenclature system is adopted to avoid confusion.
• This nomenclature was first proposed by Smith and Nattens in 1973.
• Every restriction enzyme would have a specific name which would
identify it uniquely.
• The first three letters, in italics, indicate the biological source of the
enzymes, the first letter being the initial of the genus and the second and
third being the first two letters of the species name.
15. NOMENCLATURE OF RESTRICTION ENZYMES
• Thus restriction enzymes from Escherichia coli are called Eco;
• Haemophilus influenzae becomes Hin;
• Diplocococcus pneumoniae Dpn and so on.
• Then comes a letter that identifies the strain of bacteria; Eco R for strain
R.
• Finally there is a roman numeral for the particular enzyme if there are
more than one in the strain in question;
• Eco RI for the first enzyme from E. coli R, Eco RII for the second.
16.
17. RESTRICTION SITES
• Restriction enzymes usually recognize a specific DNA sequence of 4, 5, 6 nucleotides
in length and cleave the DNA within the restriction site.
• There are 4 bases in DNA, randomly distributed.
• The expected frequency of any particular sequence can be calculated as 4 n where n
is the length of recognition sequence.
• Thus tetranucleotide sites will occur every 256 base pair, pentanucleotide sites will
occur every 1025 base pair and hexanucleotide sites will occur every 4096 base pairs.
• The complementary sequences are also known as palindrome sequences or
palindromes.
18. RESTRICTION SITES
• Recognition sites are the palindromes or palindromic sequences.
• Palindromes are the nucleotide-pair sequences that are the same when read forward
(left to right) or backward (right to left) from a central axis of symmetry i.e. two
strands are identical when both are used in the same polarity i.e. in 5’ 3’ direction.
• For example the phrase shown here reads the same in either of the directions (left to
right and right to left):
•
• ANDMADAMDNA
• 5’- GAATTC-3’
• 3’ CTTAAG-5’
19. TYPES OF RESTRICTION ENDONUCLEASES
• The restriction endonucleases can be divided into three groups as type I,
II and III.
• Types I and III have an ATP dependent restriction activity and a
modification activity resident in the same multimeric protein.
• Both these types recognize unmethylated recognition sequences in DNA.
• Type I enzymes cleave the DNA at random site, whereas Type III cleave at
a specific site.
• Type II restriction modification system possess separate enzymes for
endonuclease and methylase activity and are the most widely used for
genetic manipulation.
20. TYPE I RESTRICTION ENDONUCLEASES
• These restriction enzymes recognize the recognition site, but cleave the
DNA somewhere between 400 base pairs (bp) to 10,000 bp or 10 kbp
right or left.
• The cleavage site is not specific.
• These enzymes are made up of three peptides with multiple functions.
• These enzymes require Mg++, ATP and S adenosyl methionine for
cleavage or for enzymatic hydrolysis of DNA.
•
• These enzymes are studied for general interest rather than as useful tools
for genetic engineering.
21. TYPE I RESTRICTION ENDONUCLEASES
• These enzymes are composed of mainly three subunits, a specificity
subunit that determines the DNA recognition site, a restriction subunit,
and a modification subunit
• The recognition site is asymmetrical and is composed of two specific
portions in which one portion contain 3–4 nucleotides while another
portion contain 4–5
• EXAMPLE: EcoK
22. TYPE II RESTRICTION ENZYMES
• Restriction and modification are mediated by separate enzymes so it is
possible to cleave DNA in the absence of modification.
• Although the two enzymes recognize the same target sequence, they can
be purified separately from each other.
• Cleavage of nucleotide sequence occurs at the restriction site.
• These enzymes are used to recognize rotationally symmetrical sequence
which is often referred as palindromic sequence.
23. TYPE II RESTRICTION ENZYMES
• These palindromic binding site may either be interrupted (e.g. BstEII
recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide)
or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).
• They require only Mg2+ as a cofactor and ATP is not needed for their
activity.
• Type II endonucleases are widely used for mapping and reconstructing
DNA in vitro because they recognize specific sites and cleave just at these
sites.
24. STEPS INVOLVED
• These enzymes have nonspecific contact with DNA and initially bind to
DNA as dimmers.
• The target site is then located by a combination of linear diffusion or
“sliding” of the enzyme along the DNA over short distances, and
hopping/jumping over longer distances.
• Once the target restriction site is located, the recognition process
(coupling) triggers large conformational changes of the enzyme and the
DNA, which leads to activation of the catalytic center.
• Catalysis results in hydrolysis of phosphodiester bond and product
release.
26. BLUNT ENDED FRAGMENTS
• Blunt end cutters Type II restriction enzymes of this class cut the DNA
strands at same points on both the strands of DNA within the
recognition sequence.
• The DNA strands generated are completely base paired. Such
fragments are called as blunt ended or flush ended fragments.
27. STICKY ENDED FRAGMENTS
• Cohesive end cutter Type II restriction enzymes of this class cut the DNA
stands at different points on both the strands of DNA within the recognition
sequence.
• They generate a short single-stranded sequence at the end.
•
• This short single strand sequence is called as sticky or cohesive end.
• This cohesive end may contain 5 -PO 4 or 3 -OH, based upon the terminal
molecule (5 -PO4 or 3 -OH).
• These enzymes are further classified as 5end cutter (if 5 -PO 4 is present) or 3 -
end cutter (if3' -OH is present).
28.
29. Type III RESTRICTION ENZYMES
• The Type III enzyme is made up of two sub-units, one specifies for site
recognition and modification and the other for cleavage.
• In a reaction, it moves along the DNA and requires ATP as source of
energy and Mg++ as co-factor.
• ATPase activity is lacking in these enzymes.
• Some examples of Type III enzyme are HpaI, MboII, FokI, and the like.
They have symmetrical recognition sites and cleave DNA at specific non-
palindromic sequences.
• For cleaving double stranded DNA two sites in opposite orientation must
be present.
• One strand of double stranded DNA is cleaved about 25-27 bp away from
the recognition site which is located in its immediate vicinity.
30.
31. APPLICATIONS OF RESTRICTION ENZYMES
• In various applications related to genetic engineering DNA is cleaved by using these
restriction enzymes.
• They are used in the process of insertion of genes into plasmid vectors during gene
cloning and protein expression experiments.
• Restriction enzymes can also be used to distinguish gene alleles by specifically
recognizing single base changes in DNA known as single nucleotide polymorphisms
(SNPs).
• This is only possible if a mutation alters the restriction site present in the allele.
• Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP)
analysis for identifying individuals or strains of a particular species.
32. RESTRICTION ENZYMES
• Different restriction enzymes present in different bacteria can recognize different or
same restriction sites.
• But they will cut at two different points within the restriction site. Such restriction
enzymes are called as isoschizomers.
• Interestingly no two restriction enzymes from a single bacterium will cut at the same
restriction site.
33. DNA LIGASES
• They are also called DNA joining enzymes. DNA ligase forms a specific
type of enzyme in molecular biology, which that facilitates the joining of
the DNA strands together by catalyzing the formation of a
phosphodiester bond.
• Mertz and Davis (1972) for the first time demonstrated that cohesive
termini of cleaved DNA molecules could be covalently sealed with E. Coli
DNA ligase and were able to produce recombinant DNA molecules.
• It plays an important role in repairing single-strand breaks in duplex DNA
in living organisms, but some forms such as DNA ligase IV) may
specifically repair double-strand breaks (i.e. a break in both
complementary strands of DNA)
34. DNA LIGASES
• Single-strand breaks are repaired by DNA ligase using the complementary strand of
the double helix as a template with DNA ligase creating the final phosphodiester bond
to fully repair the DNA.
• DNA ligase has applications in both DNA repair and DNA replication.
• In addition, DNA ligase has extensive use in molecular biology laboratories for
recombinant DNA experiments.
• Purified DNA ligase is used in gene cloning to join DNA molecules together to form
recombinant DNA.
35.
36. MECHANISM OF LIGASE ACTION
• The mechanism of DNA ligase is to form two covalent Phosphodiester bonds between
3` hydroxyl ends of one nucleotide, ("acceptor") with the 5` phosphate end of another
of another ("donor").
• ATP is required for the ligase reaction, which proceeds in three steps:
• 1) adenylation (addition of AMP) of a lysine residue in the active center of the
enzyme, pyrophosphate is released;
• 2) transfer of the AMP to the 5' phosphate of the so-called donor, formation of a
pyrophosphate bond;
• 3) formation of a phosphodiester bond between the 5' phosphate of the donor and
the 3’ hydroxyl of the acceptor.
41. E.COLI DNA LIGASE
• The E.coli DNA ligase is encoded by the lig gene.
• DNA ligase in E.coli, as well as most prokaryotes, uses energy gained by
cleaving nicotinamide adenine dinucleotide (NAD) to create the
phosphodiester bond.
• It does not ligate blunt-ended DNA except under conditions of molecular
crowding with Polyethylene glycol, and cannot join RNA to DNA
efficiently.
42. T4 DNA LIGASE
• The DNA ligase from bacteriophage T4 is the ligase most commonly used in laboratory
research.
• It can ligate cohesive or “sticky ends” of DNA, oligonucleotides, as well as RNA and
RNA-DNA hybrids, but not single-stranded nucleic acids.
• It can also ligate blunt – ended DNA with much greater efficiency than E.coli DNA
ligase.
• Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it uses ATP as a
cofactor.
• Some engineering has been done to improve the in vitro activity of T4 DNA ligase.