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BLOTTING AND MICROARRAYS

SUBMITTED TO: MISS MARYUM
M
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IV

UNIVERSITY OF THE PUNJAB,
LAHORE
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HISTORY
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INTRODUCTION
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II
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IV
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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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e
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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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Samples can be in the form of tissue
culture supernatants, blood serum, cell extracts, or other preparations
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It is then
incubated with a primary antibody followed by a detection antibody or a primary antibody
conjugated to a detection molecule (commonly HRP or alkaline phosphatase)
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Dot blot is conventionally performed on a piece of nitrocellulose membrane or PVDF membrane
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Finally, for chemiluminescence imaging, the piece of membrane need to be wrapped in a
transparent plastic film filled with enzyme substrate
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For chemiluminescence signal detection, apparatus need to be dissassembled and the
membrane need to be taken out and wrapped in a transparent plastic film
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• However, these techniques have been largely—and in some cases completely—usurped by new
technologies
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• Northern blots have given way to reverse-transcription PCR, again a more sensitive and more userfriendly technique

References
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http://www
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com/open-access/types-of-blotting-
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Harbour Laboratory Press, New York; Pages 6
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58 and 7
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46 WEBSITES 1
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htm 2
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wikipedia
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http://en
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org/wiki/Far-Eastern_blotting 4
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wikipedia
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http://www
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nlm
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gov/pubmed/18079728 6
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google
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in/books?id=ZDgh_TaLzNEC&pg=PA48&lpg=PA48&dq
=blotting+techniques+history&source=bl&ots=BUsrvNI6_K&sig=I1NbWeJ5R Ux6SQ3LTo3k4aLKwg&hl=en&sa=X&ei=E4kAVL6zKIehugT39YL4BQ&ved=0CF4Q6AEwBw#
v=onepage&q=blotting%20techniques%20history&f=false 7
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wikipedia
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4
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6
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Introduction
Principle
Applications
Advantages
Limitations
Conclusion
References

Introduction
Definition:

DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached in an
organized grid fashion
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There are several
synonyms of DNA microarrays such as DNA chips, gene chips, DNA arrays, gene arrays and biochips
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The use of miniaturized microarrays for gene expression
profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a
microarray was published in 1997 by Pat Brown’s group
a) A DNA chip can be manufactured to contain hundreds of thousands of synthetic single-stranded DNA
sequences
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c) The unknown DNA is inserted into the chip and allowed to hybridize with the DNA on the chip
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The bound DNA will be
detected by its fluorescent dye and analyzed by a computer
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PRINCIPLE
The principle of DNA microarrays lies on the hybridization between the nucleotide
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The property of complementary nucleic acid sequences is to
specifically pair with each other by forming hydrogen bonds between complementary nucleotide base
pairs
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Steps in the design and implementation of a DNA microarray:
1) Probe (cDNA/oligo with known identity)
2) Chip fabrication (Putting probes on the chip)
3) Target (fluorecently labeled sample)
4) Assay
5) Readout

6) Informatics Small oligonucleotides, cDNAs, chromosome
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RNA, (mRNA) cDNA
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Fluorescenc e, probeless (conductanc e, MS, electrophore sis)
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Procedure:
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Purification
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Labelling
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Scanning 7
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cDNA based microarray 2
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– The process of measuring
gene expression via cDNA is called expression analysis
Thousand genes are simultaneously assessed – Study the effects of certain treatments, diseases, and
developmental stages on gene expression
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g
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g
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– Now, classify the types of cancer on the basis of the patterns of gene
activity in the tumor cells
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Drug Discovery
Toxicological Research – A rapid platform for the research of the impact of toxins on the cells and their
passing on to the progeny
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– These
variations, known as single nucleotide polymorphism e
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: Studies are followed to reveal, – Effects of
calorie restriction on gene expression – Obesity and high-fat diets – Genes responds to gluten and soy
protein

Microarray application in microbiology

Microbial Diagnostic Microarray – specific oligonucleotides, 35-70 nucleotides, in length are chemically
synthesized – the oligonucleotides are spotted, at defined positions, onto a glass slide to construct the
array – binding of fluorescently labelled probes to the oligonucleotides is detected
spectrophotometrically
Genes USED ON MDM? – Housekeeping genes – 16S rRNA, – 16S-23S rRNA intergenic transcribed
spacer region (ITS), – rpoB; RNA polymerase Beta – Hsp60/groEL; Heat shock protein – recA;
Recombinase A – gyrB; Gyrase Beta
Virulence genes – bacterial toxins – adhesins – important virulence factors – facilitate colonisation –
cause tissue damage

Bacteria detection
The complex interaction between a pathogen and its host is the molecular basis of infectious diseases
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Tuberculosis – Gryadunov et al : developed a biochip for detection of rifampicin-resistant and
isoniazid-resistant strains of M
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– The biochip identifies over 95% of rifampicin-resistant
and more than 80% of isoniazid-resistant M
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– The biochip has
77 gel elements and detects the 27 most-common mutations in the rpoB gene responsible for rifampicin
resistance as well as 11 mutations in the katG gene, five mutations in the promoter region of the inhA
gene, and five mutations in the intergenic regulatory region of the ahpC-oxyR genes all of which can
cause resistance to isoniazid
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Disadvantage: rare mutations or unknown mutations not detectable by the microarray probes The
newest generation of TB-biochips identifies mutations responsible for the emerging resistance of M
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Meningitis – Using the sequencing array, the scientists were able to correctly classify 45 samples that
were previously identified by conventional methods
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– resequencing microarrays provide
results in just 48 hours, much faster than traditional methods
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Virus detection:
Based on publications, microarray approaches can be summarized into four viral infection groups to
focus on: 1) respiratory diseases 2) hemorrhagic fever (HF) 3) neurotropic infection 4) HIV
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For instance, a universal microchip was developed for genotyping influenza A viruses with

two sets of oligonucleotide probes allowing viruses to be classified by the subtype of hemagglutinin (H1
- H13, H15, H16) and neuraminidase (N1 - N9)
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Based on microarrays, a detection and identification approach was designed for seven agents of the
Flaviviridae family: – yellow fever, – West Nile virus (WNV), – Japanese encephalitis, – and the dengue 1
- 4 viruses, which are causing severe human disease in tropical and subtropical areas all over the world
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– Also, a microarray comprising of 38 gene targets was developed
for the detection of several other viruses capable of causing CNS syndromes
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HIV – detect the pathogen but also can measure the amount of virus, a microarray was developed by
combining both methodologies

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