Gene cloning involves the following main steps:
1. Isolation of DNA using restriction enzymes to cut the DNA into fragments.
2. Insertion of the fragments into plasmid vectors using DNA ligase.
3. Transformation of host bacteria with the recombinant DNA plasmids.
4. Selection and amplification of bacteria containing the gene of interest using antibiotic resistance markers on the plasmid.
Gene Cloning Vectors - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Gene Cloning Vectors - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
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 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.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
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.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
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.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
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.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
The analysis of all transcripts within a cell is of essential importance. Molecular biology provides many approaches to clone RNA transcripts into cDNA. Large cDNA collections are in the public domain to serve the research community. Today, however, new high-speed sequencing methods allow a much deeper view into transcriptomes than possible by classical cloning.
DNA cloning is a technique for reproducing DNA fragments.
It can be achieved by two different approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
a vector is required to carry the DNA fragment of interest into the host cell.
This presentation deals with the introduction of Recombinant DNA Technology. The role of different enzymes. Specifically Restriction endonucleases and roles of various vectors.
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
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.
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 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.
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
4. What is gene cloning?
After a large number of cell divisions,
a colony, or clone, of identical host
cells is produced. Each cell in the
clone contains one or more copies of
the recombinant DNA molecule; the
gene carried by the recombinant
molecule is now said to be cloned.
4
8. Restriction endonucleases
Isoschizomers: Restriction enzymes with same sequence specificity and cut site
Neoschizomers: Enzymes that recognise the same sequence but cleave at different points
Star activity: under certain conditions like elevated pH or low ionic strength, RE are capable
of cleaving sequences which are similar but not identical to their defined recognition sequence
8
13. Restriction digestion
Temperature: 37oC
pH: 7.4
Ionic strength: NaCl & Mg+2 concentration
Dithiothreitol (DTT): stabilizes the enzyme and prevents its inactivation
One unit of enzyme activity: The amount of restriction enzyme in
microliters (µL) needed to completely digest (cleave) one microgram
(µg) of substrate DNA in one hour at the optimal temperature of the
enzyme in a 50-µL reaction volume.
Stopping RE activity:
Short incubation at 70oC
Phenol
EDTA
13
14. Quality control
14
Ligation can occur only if the 3′
and 5′ termini are left intact, and
only those molecules with a
perfectly restored recognition site
can be recleaved.
A normal banding pattern after
cleavage indicates that both the 3′
and 5′ termini are intact and the
enzyme preparation is free of
detectable exonucleases and
phosphatases
16. DNA ligation
DNA ligases: usually purified from E.coli bacteria that have been
infected with T4 phage. Discontinuity repair.
Sticky ends increase the efficiency of
ligation
16
17. Linkers and Adaptors
Linkers: short
pieces of double
stranded DNA,
of known
nucleotide
sequence, that
are synthesised
in the test tube. It
is blunt ended
but contains a
restriction site.
Problem with
linkers:
restriction sites
within DNA of
interest
17
18. Linkers and Adaptors
Adaptors: Short
synthetic
oligonucleotides
containing one
sticky end
Problem with
adaptors: two
adaptors could
ligate to one
another.
18
20. Vectors: vehicles for DNA
Desirable properties of a DNA vector:
1. Can independently replicate themselves and the foreign DNA
segments they carry;
2. contain a number of unique restriction endonuclease cleavage
sites that are present only once in the vector;
3. carry a selectable marker (usually in the form of antibiotic
resistance genes or genes for enzymes missing in the host cell) to
distinguish host cells that carry vectors from host cells that do not
contain a vector;
4. are relatively easy to recover from the host cell.
20
22. Plasmid
Plasmids are naturally occurring replicons in prokaryotic cells and, are stably
inherited in an extrachromosomal state.
Most plasmids exist as double stranded circular molecules.
Multiple copies per cell (relaxed plasmids) or limited number of copies per
cell (stringent plasmids).
Plasmid vectors are ≈1 to 1000 kilo base pairs in size.
22
23. Plasmid as a cloning vector
Contain replication origin (ori) sequence.
The ability to survive in normally toxic concentrations of antibiotics such as
chloramphenicol or ampicillin is often due to the presence in the bacterium of
a plasmid carrying antibiotic resistance genes. In the laboratory, antibiotic
resistance is often used as a selectable marker to ensure that bacteria in a
culture contain a particular plasmid.
Plasmid vectors are modified to contain a multiple cloning site (also called
the polylinker region) which has a number of unique target sites for
restriction endonucleases.
23
33. Transformation
Uptake of DNA by bacterial cells is called as transformation.
Most species of bacteria, including E.coli, take up only limited amounts of DNA
under normal circumstances.
In order to transform these species efficiently, the bacteria have to undergo some
form of physical and/or chemical treatment that enhances their ability to take up
DNA from the medium in which they grow. Cells that have undergone this
treatment are said to be competent.
The ice cold calcium chloride affects the cell wall and may also be responsible for
binding DNA to the cell surface. The actual uptake of DNA is stimulated by the
brief heat shock.
33
34. 1. Since bacterial species use a restriction-
modification system to degrade foreign DNA
lacking the appropriate methylation pattern,
including plasmids, then why don’t the
transformed bacteria degrade the foreign DNA?
2. Other methods of transformation?
3. Lipofection?
4. Transfection?
34
36. Insertional inactivation
Insertion of a DNA fragment into the plasmid destroys the integrity of one of
the genes present on the molecule. Recombinants can therefore be identified
because the characteristic coded by the inactivated gene is no longer displayed
by the host cells
36
44. Growth media
2XYT (1Litre)
Bacto Tryptone: 10g (supplies amino acids and small peptides)
Yeast extract: 16g (supplies nitrogen, sugars, inorganic and organic
nutrients)
NaCl: 5g (provides suitable osmotic environment for bacterial growth)
pH: 7.2
Autoclave same day and store at 2-8oC
Agar Plates: add 15g/L agar to media
Ampicillin
Stock: 100mg/ml
Working: 0.1mg/ml
44
45. Plasmid purification
Basic steps in isolation of plasmid DNA
1. A culture of bacteria is grown and then harvested
2. The cells are then broken open to release their contents
3. This cell extract is treated to remove all components except the DNA
4. Purification of plasmid DNA from total cell DNA on the basis of:
Size
Conformation
45
46. Cell disruption: chemical lysis
Lysozyme: digests polymeric compounds that give the cell wall its rigidity.
EDTA: removes magnesium ions that are essential for preserving the overall
structure of the cell envelope, and also inhibits cellular enzymes that could
degrade DNA.
Detergent (SDS/Triton X-100): aid the process of lysis by removing lipid
molecules and thereby cause disruption of the cell membrane.
Components such as partially digested cell wall fractions can be pelleted by
centrifugation, leaving the cell extract as a reasonably clear supernatant.
46
47. Plasmid purification on the basis of size
If the cells are lysed under very carefully controlled conditions, only a minimal
amount of chromosomal DNA breakage occurs. The resulting DNA fragments can
be removed with the cell debris by centrifugation.
Treatment with EDTA and lysozyme is carried out in the presence of sucrose,
which prevents the cells from bursting immediately. Instead, sphaeroplasts are
formed.
Cell lysis is now induced by adding a non-ionic detergent such as Triton X-100
(ionic detergents, such as SDS, cause chromosomal breakage).
This method causes very little breakage of the bacterial DNA, so centrifugation
leaves a cleared lysate, consisting almost entirely of plasmid DNA.
47
49. Plasmid purification on the basis of
conformation
1. Alkaline denaturation
method
2. EtBr-CsCl density
gradient centrifugation
49
50. Alkaline denaturation method
This method makes use of the observation that there is a narrow range of pH
(12-12.5) within which denaturation of linear DNA, but not covalently closed
circular DNA, occurs.
Chromosomal DNA remains in a high molecular weight form but is denatured.
Upon neutralization with acidic sodium acetate, the chromosomal DNA
renatures and aggregates to form an insoluble network.
The high concentration of sodium acetate causes precipitation of protein-SDS
complexes and high molecular weight RNA.
50
51. Alkaline denaturation method
Provided the pH of the alkaline denaturation step has been carefully controlled,
the CCC plasmid DNA molecules will remain in a native state and in solution,
while the contaminating macromolecules co-precipitate.
The precipitate can be removed by centrifugation and the plasmid concentrated
by ethanol precipitation.
If necessary, the plasmid DNA can be purified further by gel filtration.
51
52. Alkaline denaturation method
Commercially available kits take advantage of the benefits of alkaline lysis and
have as their starting-point the cleared lysate.
The plasmid DNA is selectively bound to an ion-exchange material, prepacked in
columns or tubes, in the presence of a chaotropic agent (e.g. guanidinium
hydrochloride).
After washing away the contaminants, the purified plasmid is eluted in a small
volume.
These kits improve the yield and purity of plasmid DNA.
52
55. Plasmid preparation kits by size
55
Minipreparation: rapid, small-scale isolation of plasmid DNA from bacteria. A
typical plasmid DNA yield of a miniprep is 50 to 100 µg.
Midipreparation:The starting E. coli culture volume is 15-25 mL of lysogeny
broth (LB) and the expected DNA yield is 100-350 µg.
Maxipreparation: The starting E. coli culture volume is 100-200 mL of LB and
the expected DNA yield is 500-850 µg.
Megapreparation: The starting E. coli culture volume is 500 mL – 2.5 L of LB
and the expected DNA yield is 1.5-2.5 mg.
Gigapreparation: The starting E. coli culture volume is 2.5-5 L of LB and the
expected DNA yield is 7.5–10 mg.
56. 56
If PCR is so much better, then why at all we need to perform gene cloning experiments ?
57. References & further reading
57
Gene Cloning and DNA Analysis: An Introduction. 6th edition. By T.A. Brown.
Principles Of Gene Manipulation. 6th edition. By Sandy B. Primrose, Richard M.
Twyman and Robert W. Old.
Molecular Cloning: A Laboratory manual. 3rd edition. By J. Sambrook, and
David W. Russell.