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
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
Applications of genomics and proteomics pptIbad khan
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In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
Applications of genomics and proteomics pptIbad khan
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genomics and proteomics ppt
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Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
This presentation deals with the introduction of Recombinant DNA Technology. The role of different enzymes. Specifically Restriction endonucleases and roles of various vectors.
A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms.
More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host.
Recombinant DNA molecules are produced with one of the following objectives:
1. To obtain large number of copies of specific DNA fragments.
2. Large scale production of the protein encoded by the gene.
3. Integration of the desired DNA fragment into target organism where it expresses itself.
Drought tolerant-genetically modified plants:
Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants
Of all abiotic stress, drought is regarded as the most damaging
Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management
Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success.
Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions.
Unlike conventional plant breeding there is no need of repeated back crossing
Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved.
Electrophoresis of LDH Isoenzymes and Activity StainingASHIKH SEETHY
The slides prepared for MD(Biochemistry) and MSc (Biochemistry) teaching comprehensively covers isoenzymes, isoforms, clinical utility of Lactate Dehydrogenase (LDH), LDH isoenzymes and basics of zymography.
Download and view in presenter mode for better visual experience.
Comprehensive description of various primary dyslipidemias, cholesterol transport and molecular mechanisms involved.
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Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
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A comprehensive coverage of Enzymes including basics, mechanisms of enzyme catalysis, enzyme inhibition and clinical applications, mostly based on Stryer- Biochemistry. The slides were intended for MBBS teaching, but should benefit the students of Biochemistry and allied sciences.
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A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
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Medical Technology Tackles New Health Care Demand - Research Report - March 2...pchutichetpong
M Capital Group (“MCG”) predicts that with, against, despite, and even without the global pandemic, the medical technology (MedTech) industry shows signs of continuous healthy growth, driven by smaller, faster, and cheaper devices, growing demand for home-based applications, technological innovation, strategic acquisitions, investments, and SPAC listings. MCG predicts that this should reflects itself in annual growth of over 6%, well beyond 2028.
According to Chris Mouchabhani, Managing Partner at M Capital Group, “Despite all economic scenarios that one may consider, beyond overall economic shocks, medical technology should remain one of the most promising and robust sectors over the short to medium term and well beyond 2028.”
There is a movement towards home-based care for the elderly, next generation scanning and MRI devices, wearable technology, artificial intelligence incorporation, and online connectivity. Experts also see a focus on predictive, preventive, personalized, participatory, and precision medicine, with rising levels of integration of home care and technological innovation.
The average cost of treatment has been rising across the board, creating additional financial burdens to governments, healthcare providers and insurance companies. According to MCG, cost-per-inpatient-stay in the United States alone rose on average annually by over 13% between 2014 to 2021, leading MedTech to focus research efforts on optimized medical equipment at lower price points, whilst emphasizing portability and ease of use. Namely, 46% of the 1,008 medical technology companies in the 2021 MedTech Innovator (“MTI”) database are focusing on prevention, wellness, detection, or diagnosis, signaling a clear push for preventive care to also tackle costs.
In addition, there has also been a lasting impact on consumer and medical demand for home care, supported by the pandemic. Lockdowns, closure of care facilities, and healthcare systems subjected to capacity pressure, accelerated demand away from traditional inpatient care. Now, outpatient care solutions are driving industry production, with nearly 70% of recent diagnostics start-up companies producing products in areas such as ambulatory clinics, at-home care, and self-administered diagnostics.
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One of the most developed cities of India, the city of Chennai is the capital of Tamilnadu and many people from different parts of India come here to earn their bread and butter. Being a metropolitan, the city is filled with towering building and beaches but the sad part as with almost every Indian city
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
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Restriction Digestion and its Applications
1. 1
RESTRICTION DIGESTION AND ITS APPLICATIONS
Endonucleases cleave internal phosphodiester bonds in polynucleotides, while exonucleases cleave
polynucleotides from their ends. Restriction endonucleases are group of enzymes that cleave DNA at or
near a specific sequence.
Evolutionarily, their role is to protect bacteria from invading foreign DNAs and that is why they are
endowed with exquisite sequence specificity.
There are 4 types of restriction endonucleases:
Type I enzymes are complex, multisubunit enzymes that cut DNA at random sites far from their
recognition sequences.
Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They
produce discrete restriction fragments and distinct gel banding patterns, and they are the only class
used in the laboratory for routine DNA analysis and gene cloning.
Type III enzymes also cleave outside of their recognition sequences and require two such sequences in
opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete
digests.
Type IV enzymes recognize modified DNA, usually, methylated DNA
Restriction endonucleases are named based on the bacterial strain from which they are isolated. Example:
EcoRI is named after Escherichia coli RY13 strain and was the first (I) restriction endonuclease to be
isolated from this strain.
Digestion of the DNA by a restriction endonuclease can lead to generation of digested products either with
flushed ends (blunt ends) or with overhangs (sticky ends).
Because of the ability of Type II enzymes cut DNA at defined positions close to or within their recognition
sequences, they have applications in various molecular biology techniques, as detailed below:
1. MOLECULAR CLONING:
Molecular cloning refers to the isolation of a DNA sequence from any species (often a gene), and its
insertion into a vector for propagation, without alteration of the original DNA sequence. Plasmids are the
most commonly used vectors are can accept up to 10 kilobase pairs of foreign DNA.
2. 2
The human diploid genome is composed of 6 billion base pairs and isolation of a specific gene to be inserted
into a vector is a laborious process. Hence, the source of the gene to be inserted into the vector can be (i) a
gene library, in which the clones containing the gene of interest have already been identified or (ii)
complementary DNA (cDNA) obtained from mRNA of a cell expressing the gene in large quantities. The
cDNA can be amplified using a polymerase chain reaction with primers specific for the gene of interest and
the amplicon obtained can be used for cloning.
Both the plasmid and the DNA to be inserted are digested by the same restriction enzyme(s). Usually,
restriction enzymes that produce sticky ends are used. This will lead to generation of 5’ and 3’ overhangs
in the plasmid and the insert, which are complementary to each other, as shown in the diagram:
3. 3
The insert and the plasmid can then be ligated using DNA ligase in the presence of ATP, to generate the
recombinant plasmid.
Note in the above diagram that the plasmid also contains various other sequences:
a. Origin: Refers to the sequence for origin of replication, which is necessary for multiplication of plasmid
in the host organism.
b. Plasmid also contains one or more markers that enable us to differentiate between the host organisms
in which the plasmid is present, compared to those which do not have the plasmid. Example: Presence
of an antibiotic resistance gene enables bacteria containing the plasmid to grow in a growth medium
containing that particular antibiotic.
c. MCS or Multiple Cloning Site, aka polylinker is a feature of engineered plasmids and contains multiple
restriction sites close to each other, and thus provide versatility in the DNA fragments that can be
inserted.
Once the recombinant plasmid has been made after the ligation of restriction-digested plasmid and insert,
the recombinant plasmid is introduced into a bacteria. The process by which foreign DNA is introduced
into a cell is known as transformation. A bacterial cell can be transformed by any of the following
methods:
1. Incubating the bacterial cells under cold conditions in presence of a divalent cation (eg.Ca++
) along with
the plasmid, followed by a heat shock. The divalent cations generate coordination complexes with the
negatively charged DNA molecules, facilitating their entry into the cell.
2. Electroporation in which the cells are briefly shocked with an electric pulse. This leads to transient pore
formation in the plasma membrane, through which the plasmid can enter the cell; the pore is later
repaired by various repair mechanisms.
The bacteria that is transformed are then grown in the presence of appropriate marker, so as to select the
colonies containing the plasmid. These colonies are then used to isolate the plasmid in a procedure that is
analogous to genomic DNA isolation along with a few modifications.
APPLICATIONS OF MOLECULAR CLONING:
1. Production of recombinant proteins: Recombinant vector that contains the coding sequence of a
particular protein of interest under suitable promoter can be introduced into appropriate prokaryotic or
eukaryotic vector to express proteins in larger amounts. Examples of recombinant proteins include
various drugs like insulin, erythropoietin, clotting factors etc. and vaccines like hepatitis B vaccine
which contains HBsAg (Hepatitis B surface Antigen).
2. Production of DNA library: DNA library is a collection of DNA fragments that have been cloned into
vectors so that researchers can identify and isolate the DNA fragments that interest them for further
study. There are basically two kinds of libraries: genomic DNA and cDNA libraries.
3. Site directed mutagenesis: The sequence of a protein can be altered at specific amino acid residues by
mutating the specific codons in the coding sequence which can be then cloned and expressed. This is
helpful in understanding the pathogenesis of genetic diseases, mapping the active site amino acids of
enzymes etc.
4. Gene therapy: Preparation of lentiviral/adenoviral vectors with the correct insert coding for the protein
to be expressed involves molecular cloning.
RESTRICTION MAPPING:
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. After a DNA segment has been digested using a restriction
4. 4
enzyme, the resulting fragments can be examined agarose gel electrophoresis, which separates the
fragments of DNA generated according to their size.
One common method for constructing a restriction map involves digesting the unknown DNA sample in
three ways. Here, two portions of the DNA sample are individually digested with different restriction
enzymes, and a third portion of the DNA sample is double-digested with both restriction enzymes at the
same time. Next, each digestion sample is separated using gel electrophoresis, and the sizes of the DNA
fragments are recorded. The total length of the fragments in each digestion will be equal. However, because
the length of each individual DNA fragment depends upon the positions of its restriction sites, each
restriction site can be mapped according to the lengths of the fragments. The final representation of the
DNA segment that shows the positions of the restriction sites is called a restriction map.
2. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP):
RFLP refers to the presence of DNA fragments of different lengths, after digestion of homologous DNA
samples in question with specific restriction endonucleases. In RFLP analysis, the DNA fragment is
digested by one or more restriction endonucleases; depending on the presence or absence of the restriction
sites for the specific restriction endonucleases, the nucleotide changes in the DNA can be profiled.
Let us see an example:
The genetic defect in sickle cell anemia is a point mutation (transversion) where T is substituted for A in
the coding region of beta hemoglobin gene. This substitution results in a change in the codon from GAG
(glutamic acid) to GUG (valine).
The figure below shows the restriction site of MstII corresponding to 6th
position of beta globin:
5. 5
Beta globin gene has 3 restriction sites for MstII.
One of these restriction sites is lost in sickle cell anemia.
Thus the
restriction pattern will be different in a DNA sample from a normal individual, compared to those with
sickle cell trait or sickle cell disease.
This can be detected using:
1. Sequence specific probes against these fragments using a Southern Blot. In Southern Blot:
DNA is digested with a set of restriction enzymes
The fragments are separated based on their molecular weight (size) using gel electrophoresis
The DNA is transferred to a polymer membrane (usually nitrocellulose or nylon) from the gel. This
process is known as blotting. Blotting helps to prevent the diffusion of DNA which can occur in a gel.
Moreover, blots are less fragile, compared to a gel.
Sequence specific labelled nucleotide oligomers (also known as probes) are incubated with the blot.
The probes are radiolabelled or fluorescent labelled. The probes will bind only to their complementary
DNA sequence in the blot (hybridization).
The DNA fragment of interest can be detected by exposing the blot to an X-ray film (in case of
radiolabelled probes) or by examining under a fluorescence microscope (in case of fluorescent labelled
probes).
6. 6
The below diagram shows MstII restriction pattern of beta globin gene in sickle cell trait, sickle cell disease
and a normal individual, when detected using Southern blotting:
1. PCR-RFLP:
In PCR-RFLP, PCR is performed to amplify a particular region of DNA and the PCR products are subjected
to digestion using restriction endonucleases. Since the sequence of interest in the DNA is amplified to
signifance amounts, the products can be visualised under UV light after an agarose gel electrophoresis, thus
avoiding the radioactive hazards that are involved in Southern blotting.
The diagram given in the next page explains the principle of PCR-RFLP in diagnosis of sickle cell anemia
Note that RFLP techniques are NOT frequently used for genotyping at present. The preferred method is
DNA sequencing. Can you think why?
7. 7
3. DNA FINGERPRINTING:
In DNA fingerprinting, genomic DNA is subjected to digestion by a specific set of restriction
endonucleases. Humans share a genome sequence similarity of 99.9%. This means DNA sequence of two
individuals differs in 1 in every 1000 nucleotides. Some of these sites that differ may be restriction sites.
So the restriction pattern of DNA from two individuals generally differ. This is another example of RFLP.
DNA fingerprinting was used to establish a link between a biological evidence and a suspect in a criminal
investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If
the two DNA profiles are a match, then the evidence came from that suspect. Conversely, if the two DNA
profiles do not match, then the evidence cannot have come from the suspect.
Since RFLPs are inherited, DNA fingerprinting were also used to establish paternity.
The procedure is almost same as the RFLP analysis using Southern Blot which we discussed earlier, except
for the fact that multiple enzymes are used here, and different samples are compared.
Nowadays, RFLP has been replaced by analysis of STR (short tandem repeats) in criminalistics.
PROTOCOL FOR RESTRICTION DIGESTION OF PLASMID:
You have been provided with the following:
1. 4 µg of plasmid DNA
2. Restriction enzymes A and B (20 Units each)
3. Buffer for restriction enzyme
4. Nuclease free water
5. 1% Agarose gel (containing ethidium bromide)
6. Gel loading dye
7. DNA ladder (molecular weight marker)
8. PCR tubes (0.2 mL)
8. 8
Make the reaction mixtures as shown below:
Sl No Component Tube A Tube B Tube C
1. Plasmid 1 µg 1 µg 1 µg
2. Nuclease free water Upto 50 µL Upto 50 µL Upto 50 µL
3. Buffer 5 µL 5 µL 5 µL
4. Enzyme A 10 Units - 10 Units
5. Enzyme B - 10 Units 10 Units
1. Mix components by pipetting the reaction mixture up and down. Follow with a quick spin-down in a
microcentrifuge.
2. Incubate at 37 ⁰C for 15 minutes.
3. Add 10 µL of gel loading dye to each tube.
4. Also, prepare tube D by adding 1 µg DNA to 1 µL gel loading dye.
5. Load the contents of the tube into the agarose gel, along with molecular weight marker.
6. Perform agarose gel electrophoresis (80 V for 60 minutes) and visualise your gel under uv
transilluminator/ gel documentation system.
7. Based on your findings, create a restriction map of the plasmid.
PRECAUTIONS:
1. Wear gloves while performing all the steps.
2. Restriction enzyme should be added as the last component of the reaction mixture
3. Restriction enzymes should be always kept on ice
4. Use UV safety measures while visualising the gel.