This document discusses various methods for ligating DNA fragments, including blunt end ligation, sticky end ligation using linkers or adaptors, and homopolymeric tailing. Blunt end ligation is less efficient than sticky end ligation. Linkers and adaptors are oligonucleotides used to create sticky ends for ligation, while homopolymeric tailing uses terminal transferase to add homopolymer tails to blunt ends before ligation. The goal is to efficiently join vector and insert DNA fragments for recombinant DNA construction.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
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
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.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
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
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.
Recombination in repair n damage of DNA.pptxANAKHA JACOB
• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.
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.
DNA replication, repair and recombination NotesYi Fan Chen
DNA, replication, repair and recombination Notes based on Molecular biology of the cell. Biology Elite: biologyelite.weebly.com, please use together with the presentation
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
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.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Richard's aventures in two entangled wonderlandsRichard 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.
3. LIGATION
• Construction of a recombinant
DNA molecule- joining together
of the vector and DNA to be
cloned
• enzyme that catalyzes the
reaction is called DNA ligase.
Ligation: the final step in construction
of a recombinant DNA molecule.
4. DNA LIGASE
• cellular enzyme- function is to
repair broken phosphodiester
bonds that may of DNA
replication or recombination or
repairing.
• The first DNA ligase was purified
and characterized in1967
5. BLUNT END LIGATION
• Ligation- 2 blunt ended
fragments
• Not very efficient
• Ligase is unable to “catch hold”
of the molecule
• Performed at high
concentrations DNA and ligase
T.A. BROWN, Gene Cloning and DNA
Analysis, Sixth Edition , pg-64
6. STICKY END LIGATION
• Ligation - 2 sticky ended
fragments
• Ligation is more efficient
• base pair with one another by
hydrogen bonding
• annealing of complementary
overhangs brings 5’P and 3’OH
into close proximity
The different joining reactions catalyzed by DNA ligase: (a)
ligation of blunt-ended molecules; (b) ligation of sticky-ended
molecules.
8. LINKERS
• Chemically synthesized ds DNA
oligonucleotides
• It is blunt ended
• Contain one or more restriction
sites for cleavage e.g., Eco RI, Hind
III, Bam HI, etc
• ligated to blunt end DNA by using
DNA ligase
Figure 4.21 Linkers and their use: (a) the structure of a typical linker; (b) the attachment of
linkers to a blunt-ended molecule.
9. • linker- ligation mixture at high
concentration to form a chain
structure
• Bam H1 cleaves at recognition
site
• Cleaved linkers Carry Bam H1
sticky ends
• Drawback- DNA fragment sometimes already
possesses the restriction sites for producing
cohesive ends.
Figure 4.22 A possible problem with the use of
linkers. Compare this situation with the desired
result of BamHI restriction, as shown in Figure
4.21(b).
11. ADAPTORS
• short synthetic oligonucleotides
with performed cohesive ends
• Sticky end pair - Dimers
• Ends of adaptors is chemically
modified
Figure 4.23 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.
12. figure 4.24 The distinction between the 5′ and 3′ termini of a
polynucleotide.
Chapter 4 Manipulation of Purified DNA
13. • 3′-OH terminal the same, 5′-P is
modified to 5′-OH terminus
• Alkaline phosphatase to prevent
self ligation.
• treated with Polynucleotide
kinases to produce 5’-P sticky
ends
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.
14. HOMOPOLYMERIC TAILING
• Technique at which sticky ends can be
produced on a blunt ended DNA
• Polymer – all subunits are same ex-
polydeoxyguanosine
• Tailing – terminal deoxynucleotidyl
transferase
Recombinant RNA Technology Restriction Enzyme
Homopolymer tailing Biotechnology | Applied
Biosciences.
15. Figure 4.26 Homopolymer tailing: (a) synthesis of a
homopolymer tail; (b) construction of a recombinant
DNA molecule from a tailed vector plus tailed insert
DNA
16. • But when tails are not same
length- nicks and discontinuities
• Klenow polymerase- to fill in the
nicks
• DNA ligase - synthesize the
phosphodiester bonds.
Source: (c) repair of the recombinant DNA molecule
Part I The Basic Principles of Gene Cloning and DNA Analysis
17. CONCLUSION
• To produce cohesive ends in vector and insert DNA
• molecules are used to ligate gene of interest with cohesive end
vectors
• used to add sicky ends to cDNA allowing it to be ligated into
the plasmid much more efficiently
18. REFERENCE
• T.A. BROWN, Gene Cloning and DNA Analysis, Sixth Edition, Chapter
4, 53-69.
• S.B.Primrose and R.M.Twyman, Principle of gene manipulation and
genomics, seventh edition, chapter 3, 44-49.
• https://recombinant-dna-cloning-technology-chp-7-dna-cloning
• https://www.ecarepk.com/2020/09/rDNA-restriction-enzyme-DNA-
ligase-linker-adapter-homopolymer-tailing.html
