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BLOTTING AND MICROARRAYS

SUBMITTED TO: MISS MARYUM
M
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UNIVERSITY OF THE PUNJAB,
LAHORE
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HISTORY
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INTRODUCTION
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II
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SOUTHERN BLOTTING
NORTHERN BOLTTING
WESTERN BLOTTING -Far Western Blotting
EASTERN BLOTTING
DOT BLOT

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REFERENCES

HISTORY
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1966-1963-Nygaard and Hall: Shown that single stranded DNA can be immobilized on
nitrocellulose filters
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1970s: Possibility of mapping whole genomes arose along with the era of rDNA and gene
cloning
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1965-Denhardt and Spiegelman: Nucleic acid thus fixed can be detected with exquisite
sensitivity by hybridization to radiolabelled probes
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Harry Towbin gave faster and simpler approach
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Neal Burnette’s (1981) technique named Western Blottingcolleagues: Detection of RNA
(Northern Blotting)

DNA HYBRIDIZATION
DNA hybridization involves the formation of a double-stranded nucleic acid, either a DNA double
helix or an RNA–DNA duplex
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Hybridization analysis makes use of the unique linear array of
bases, which in one strand specifies a unique complementary strand
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Thus, a 15- to 21-nucleotide (nt) single DNA strand will only hybridize to a unique
complementary single region of the entire 3 billion bases in human DNA
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This membrane may be nitrocellulose PVDF or nylon membrane
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But sometimes it can be done by directly
transferring the molecules onto the membrane
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Examples: Ethidium bromide, Crystal violet, Safranine and Ossmium
tetroxide etc

TYPES
1)Southern blotting:
Southern blotting is named after Edward M
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This method is used for analysis of DNA
sequences
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• Then, these fragments are electrophoresed on separating gel so that they can separate according to
their size
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• After separating these fragments, placed a nitrocellulose sheet over the separating gel
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• After that the membrane is exposed to ultraviolet radiation so that the fragments are permanently
attached to the membrane
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But the DNA probe is labeled so that it can
easily detect, when the molecule is tagged with a chromogenic dye
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Applications
i) It is used in the technique called RFLP (Restriction fragment length polymorphism) mapping
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iii) To identify the gene rearrangements
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Neal Burnette
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It involves the following steps:
• Firstly, isolating the protein from particular sample
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• Then, protein- SDS complex is placed on top of the gel in the well
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After that the
samples are added in the remaining wells
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Protein is pulled down
to the positive pole of the well because it is tightly bound to SDS which is negatively charged
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• After this step, gel is placed against a membrane and current is passed across the gel so that all the
proteins are transferred onto the membrane
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In this method, firstly block the membrane with non-specific
protein in order to prevent antibody from binding to the membrane where the protein is not present
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These antibodies are responsible for recognizing
a specific amino-acid sequence
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• Now these antibodies are conjugated with an enzyme and recognize the primary antibody
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• Here, chemiluminescent substrate is used for detection
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Applications:
i) Used in clinical purposes
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iii) Used to quantifying a gene product
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This method is used to analyse and detection of RNA in a sample
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• Separate these RNA on agarose gels containing formaldehyde as a denaturing agent for the RNA
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• Then transfer these RNA fragments onto the carrier membrane i
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• After transferring the RNA,it is fixed to the membrane by using UV or heat
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Wash off the unbound probe and at the end mRNA-DNA hybrid are then detected by X-ray film
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4) Eastern blotting
Eastern blotting is given by Bogdanov
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Mostly blotted proteins after transferring onto the membrane are analyzed
for PTMs by using a probe and hence identify carbohydrates and lipids
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• Then, these separated molecules are transferred horizontally on the nitrocellulosic membrane
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These antibodies are responsible for recognizing
a specific amino-acid sequence
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• These labelled probes confirm the molecule of interest
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ii) Used for binding studies by using various ligand
iii) Used to purify various phospholipids
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It represents a
simplification of the western blot method, with the exception that the proteins to be detected are not
first separated by electrophoresis
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The technique offers significant savings in time, as chromatography or gel electrophoresis, and
the complex blotting procedures for the gel are not required
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Performing a dot blot is similar in idea to performing a western blot, with the advantage of
faster speed and lower cost
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b) Unknown DNA from a patient is separated into single strands, enzymatically cut and labeled with a
fluorescent dye
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d) The tagged DNA will bind only to the complementary DNA on the chip
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The red light is a gene expressed in normal
cells; green is a mutated gene expressed in tumor cells; and yellow, in both cells
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Using this
technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be
screened in a single hybridization
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By using an array containing many DNA samples, scientists can determine, in a single experiment, the
expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA
bound to each site on the array
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Photolithogra --phy, pipette, piezoelectric
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Hybridization
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Robotics control, Image processing, DBMS,bioinf ormatics
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Sample preparation 2
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Reverse Transcription 4
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Hybridization 6
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Normalization and analysis

There are 2 types of DNA Chips/Microarrays:
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Oligonucleotide based microaaray

APPLICATIONS
Gene expression analysis – Not all the genes in the human genome are active at all times – used to
detect DNA , or detect RNA that may or may not be translated into proteins
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– E
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: identify genes expression changes due to pathogens or
other organisms by comparing with uninfected cells or tissues
Help to investigate about different diseases – E
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: Earlier cancers classified on the basis of the organs in
which the tumors develop
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– Help to produce very effective drugs Disease diagnosis
Extensive application in Pharmacogenomics – Comparative analysis of the genes – Help the
identification of the specific proteins produce by diseased cells – Information used to synthesize drugs
which combat with these proteins and reduce their effect
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– Important for Toxicogenomic studies
Small microarrays to check IDs of organisms in food and feed (like GMO) and mycoplasmas in cell culture
– Mostly combining PCR and microarray technology Gene ID
Nutrigenomic research Study variations in the genes related to the influence of diets
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Microarray technology is a powerful tool to investigate the crosstalk between a pathogen and the host
as it assesses whole genome expression profiles in response to disease
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tuberculosis
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tuberculosis strains in sputum samples
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sensitivity of 80% and a specificity of 100% compare to traditional testing for rifampicin resistance
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tuberculosis so the highly effective second-line fluoroquinolone antibiotics can be administered
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But more importantly, was able to classify 12
previously unclassifiable samples into existing meningitis serotypes
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– The meningitis resequencing array can
now be used to quickly identify new meningitis strains, as well as for epidemiological studies and vaccine
research
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Respiratory Diseases Detection of etiological agents : – Some influenza microarrays are designed to
detect DNA
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Hemorrhagic Fever – Viruses associated with hemorrhagic fever (HF) are mainly found in the families
Arenaviridae, – Bunyaviridae, – Flaviviridae – Filoviridae
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Neurotropic virus – A DNA microarray for the detection of 13 specific pathogens in meningitis and
encephalitis cases was developed for the most common neurotropic viruses including HSV-1, varicellazoster virus [VZV], and enteroviruses
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This concluded that clinical sensitivity,specificity, and negative and positive predictive values of the
assay were 93%, 100%, 100%, and 83% respectively,comparing microarray to the single-virus PCR
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The study described an original approach for simultaneous quantitative
identification of these viruses in blood plasma specimens using real-time PCR with primers immobilized
o

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