Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 30 Analysis Of Gene Expression Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
ANALYSIS OF GENE EXPRESSION
1
Objectives
? Determination of RNA level
? Northern blot
? Microarrays
? Analysis of proteins
ELISA
Western blot
? Gene Sequencing and application
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ANALYSIS OF GENE EXPRESSION
Determination of RNA levels
Northern blots
Microarrays
Analysis of proteins
Enzyme-linked immunosorbent assays (ELISA)
Western blots
Proteomics: The study of all proteins expressed by a genome,
including their relative abundance, distribution,
posttranslational modifications, functions, and interactions
with other macromolecules, is known as proteomics.
Proteomics offer the potential of identifying new disease
markers and drug targets.
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Northern Blot
? The northern blot technique was developed in
1977 by James Alwine, David Kemp and George
Stank at Stanford University
? Principle:
? Northern blotting involves the use of
electrophoresis to separate RNA samples by size
and detection with a hybridization probe
complementary to part of or the entire target
sequence.
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Procedure of Northern Blotting
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? Limitations:
? Northern Blotting using radioactive probes is
very sensitive, but very time-consuming.
Northern blotting is not practical in large
clinical studies to detect the expression of
hundreds of miRNAs and it also requires large
amounts (5?25 g) of total RNA from each
sample
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Microarray
analysis
of gene
expression
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Microarray
analysis
of gene
expression
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Gene Sequencing
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". . [A] knowledge of sequences could contribute much
to our understanding of living matter."
Frederick Sanger
10
History
? The first method for determining DNA sequences
involved a location-specific primer extension strategy
established by Ray Wu in 1970
? The first DNA fragment to be sequenced belonged to
T4 bacteriophage
? In the mid-1975, Frederick Sanger and Aln Coulson
sequenced by using a plus-minus system for running
a sequencing reaction.
? .
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History contd
? Maxam and Gilbert further modified this method by
using radiolabeled DNA and chemicals (such as
hydrazine)
? One of the biggest breakthroughs in this field was
the development of chain-termination technology
using modified nucleotides by the Sanger lab in 1977
? In 1983, polymerase chain reaction (PCR) for
amplifying stretches of DNA was discovered.
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Type of gene sequencing
? First generation DNA sequencing
? Sanger sequencing
? Maxam Gilbert sequencing
? Automated DNA sequencing
? Shot Gun sequencing
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Sequencing of DNA by
the chain termination method
devised by Sanger
Maxam and Gilbert
sequencing of DNA
16
Automated DNA sequencing
? PCR used for making sequencing templates
? Fluorescently labelled ddNTPs are used
? Capillary electrophoresis
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Automated
fluorescent sequencing
detection
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? Second-generation DNA sequencing
: (Next generation sequencing)
? Pyrosequencing
? Sequencing by synthesis
? Sequencing by ligation
? Ion semiconductor sequencing
19
? Third-generation DNA sequencing
? Real-time, single-molecule sequencing
? capable of sequencing single molecules,
negating the requirement for DNA
amplification shared by all previous
technologies.
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Application of DNA sequencing
? Forensics:
?
To identify the individual
? Medicines
? To detect genes associated with some hereditary or acquired diseases
? E,g. Huntingtons disease
? CAG codon in exon 1
? Fragile X syndrome
? CGG >200
? Myotonic Dystrophy
? CUG >100
? Agriculture
? Mapping and sequencing of whole genome of microorganisms helps in making them useful for
foods or crops
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MCQ 1
? Which of the following is not an exclusively
DNA sequencing method?
? 1. Sangers
? 2. Maxam Gillbert
? 3. Edman
? 4. LMPCR (Ligation mediated PCR)
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LMPCR (Ligation mediated PCR)
? (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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MCQ2
? The sample in Sangers method after reaction
separated in
? 1. AGE
? 2. PAGE
? 3.PFGE (Pulse field gel electrophoresis)
? 4. 2-D gel electrophoresis
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MCQ3
? If a hypothetical peptide has the sequence Phe-
Tyr-Met-Pro-His.
? Calculate number of possible nucleotide
sequences.
? A. 11
? B. 8
? C. 22
? D. 32
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This post was last modified on 05 April 2022