? Determination of RNA level
? Northern blot
? Microarrays
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? Analysis of proteinsELISA
Western blot
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? Gene Sequencing and application2
ANALYSIS OF GENE EXPRESSION
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Determination of RNA levelsNorthern blots
Microarrays
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Analysis of proteins
Enzyme-linked immunosorbent assays (ELISA)
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Western blotsProteomics: The study of all proteins expressed by a genome,
including their relative abundance, distribution,
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posttranslational modifications, functions, and interactions
with other macromolecules, is known as proteomics.
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Proteomics offer the potential of identifying new diseasemarkers and drug targets.
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Northern Blot
? The northern blot technique was developed in
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1977 by James Alwine, David Kemp and GeorgeStank at Stanford University
? Principle:
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? Northern blotting involves the use of
electrophoresis to separate RNA samples by size
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and detection with a hybridization probecomplementary to part of or the entire target
sequence.
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4
Procedure of Northern Blotting
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? Limitations:
? Northern Blotting using radioactive probes is
very sensitive, but very time-consuming.
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Northern blotting is not practical in large
clinical studies to detect the expression of
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hundreds of miRNAs and it also requires largeamounts (5?25 g) of total RNA from each
sample
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6
Microarray
analysis
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of gene
expression
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7Microarray
analysis
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of gene
expression
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8Gene Sequencing
9
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". . [A] knowledge of sequences could contribute muchto our understanding of living matter."
Frederick Sanger
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10
History
? The first method for determining DNA sequences
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involved a location-specific primer extension strategy
established by Ray Wu in 1970
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? The first DNA fragment to be sequenced belonged toT4 bacteriophage
? In the mid-1975, Frederick Sanger and Aln Coulson
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sequenced by using a plus-minus system for running
a sequencing reaction.
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? .11
History contd
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? Maxam and Gilbert further modified this method by
using radiolabeled DNA and chemicals (such as
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hydrazine)? One of the biggest breakthroughs in this field was
the development of chain-termination technology
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using modified nucleotides by the Sanger lab in 1977
? In 1983, polymerase chain reaction (PCR) for
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amplifying stretches of DNA was discovered.12
Type of gene sequencing
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? First generation DNA sequencing? Sanger sequencing
? Maxam Gilbert sequencing
? Automated DNA sequencing
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? Shot Gun sequencing13
Sequencing of DNA by
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the chain termination methoddevised by Sanger
Maxam and Gilbert
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sequencing of DNA
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Automated DNA sequencing
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? PCR used for making sequencing templates
? Fluorescently labelled ddNTPs are used
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? Capillary electrophoresis17
Automated
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fluorescent sequencing
detection
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18? Second-generation DNA sequencing
: (Next generation sequencing)
? Pyrosequencing
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? Sequencing by synthesis? Sequencing by ligation
? Ion semiconductor sequencing
19
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? Third-generation DNA sequencing
? Real-time, single-molecule sequencing
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? capable of sequencing single molecules,negating the requirement for DNA
amplification shared by all previous
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technologies.
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Application of DNA sequencing
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? Forensics:
?
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To identify the individual? Medicines
? To detect genes associated with some hereditary or acquired diseases
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? E,g. Huntingtons disease
? CAG codon in exon 1
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? Fragile X syndrome? CGG >200
? Myotonic Dystrophy
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? CUG >100
? Agriculture
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? Mapping and sequencing of whole genome of microorganisms helps in making them useful forfoods or crops
21
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MCQ 1
? Which of the following is not an exclusively
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DNA sequencing method?? 1. Sangers
? 2. Maxam Gillbert
? 3. Edman
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? 4. LMPCR (Ligation mediated PCR)22
LMPCR (Ligation mediated PCR)
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? (1) primary DNA nucleotide sequences? (2) cytosine methylation patterns
? (3) DNA lesion formation and repair, and
? (4) in vivo protein-DNA footprints
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23MCQ2
? The sample in Sangers method after reaction
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separated in
? 1. AGE
? 2. PAGE
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? 3.PFGE (Pulse field gel electrophoresis)? 4. 2-D gel electrophoresis
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MCQ3
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? If a hypothetical peptide has the sequence Phe-
Tyr-Met-Pro-His.
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? Calculate number of possible nucleotidesequences.
? A. 11
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? B. 8
? C. 22
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? D. 3225