Download MBBS Biochemistry PPT 30 Analysis Of Gene Expression Lecture Notes

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

2
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.

3

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.

4
Procedure of Northern Blotting

5

? 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

6
Microarray

analysis

of gene

expression

7

Microarray

analysis

of gene

expression

8
Gene Sequencing

9

". . [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.

? .

11

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.

12
Type of gene sequencing

? First generation DNA sequencing

? Sanger sequencing
? Maxam Gilbert sequencing
? Automated DNA sequencing
? Shot Gun sequencing

13
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

17

Automated

fluorescent sequencing

detection

18
? 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.

20
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

21

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)

22
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

23

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

24
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

25

This post was last modified on 05 April 2022