Gene regulation, History and Evolution , Traditional Methods: Northern blot quantitative reverse transcription PCR (qRTPCR) serial analysis of gene expression(SAGE) and DNA microarrays. DNA Chip

1. FUNCTIONAL GENOMICS & PROTEOMICS
Submitted By:
Dhaval Chaudhary
M.V.Sc. (Animal Genetics & Brreding)
Methods to Study Gene Expression

2. What is Gene Expression?
• Gene expression is the process by which information from a gene is
used in the synthesis of a functional gene product that enables it to
produce end products, protein or non-coding RNA, and ultimately
affect a phenotype, as the final effect.
• These products are often proteins, but in non-protein-coding genes
such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the
product is a functional non-coding RNA.

3. • In genetics, gene expression is the most fundamental level at which
the genotype gives rise to the phenotype, i.e. observable trait. The
genetic information stored in DNA represents the genotype, whereas
the phenotype results from the "interpretation" of that information.
• Such phenotypes are often expressed by the synthesis of proteins
that control the organism's structure and development, or that act as
enzymes catalyzing specific metabolic pathways.
• All steps in the gene expression process may be modulated
(regulated), including the transcription, RNA splicing, translation and
post-translational modification of a protein.

5. The process of gene expression involves two
main stages:
• Transcription: the production of messenger RNA (mRNA) by the
enzyme RNA polymerase, and the processing of the resulting mRNA
molecule
• Translation: the use of mRNA to direct protein synthesis, and the
subsequent post-translational processing of the protein molecule.
• Some genes are responsible for the production of other forms of RNA
that play a role in translation, including transfer RNA (tRNA) and
ribosomal RNA (rRNA).

6. • A structural gene involves a number of different components:
• Exons: Exons code for amino acids and collectively determine the
amino acid sequence of the protein product. It is these portions of
the gene that are represented in final mature mRNA molecule.
• Introns: Introns are portions of the gene that do not code for amino
acids, and are removed (spliced) from the mRNA molecule before
translation.

7. Control regions:
• Start site - A start site for transcription.
• A promoter - A region a few hundred nucleotides 'upstream' of the gene (toward the 5'
end). It is not transcribed into mRNA, but plays a role in controlling the transcription of
the gene. Transcription factors bind to specific nucleotide sequences in the promoter
region and assist in the binding of RNA polymerases.
• Enhancers - Some transcription factors (called activators) bind to regions called
'enhancers' that increase the rate of transcription. These sites may be thousands of
nucleotides from the coding sequences or within an intron. Some enhancers are
conditional and only work in the presence of other factors as well as transcription
factors.
• Silencers - Some transcription factors (called repressors) bind to regions called 'silencers'
that depress the rate of transcription.

8. Transcription
• Transcription is the process of RNA synthesis, controlled by the
interaction of promoters and enhancers. Several different types of
RNA are produced, including messenger RNA (mRNA), which
specifies the sequence of amino acids in the protein product, plus
transfer RNA (tRNA) and ribosomal RNA (rRNA), which play a role in
the translation process.

9. Transcription involves four steps:
• Initiation. The DNA molecule unwinds and separates to form a small open complex. RNA
polymerase binds to the promoter of the template strand (also known as the 'sense
strand' or 'coding strand'). The synthesis of RNA proceeds in a 5' to 3' direction, so the
template strand must be 3' to 5'.
• Elongation. RNA polymerase moves along the template strand, synthesising an mRNA
molecule. In prokaryotes RNA polymerase is a holoenzyme consisting of a number of
subunits, including a sigma factor (transcription factor) that recognises the promoter. In
eukaryotes there are three RNA polymerases: I, II and III. The process includes a
proofreading mechanism.
• Termination. In prokaryotes there are two ways in which transcription is terminated. In -
dependent termination, a protein is responsible for disrupting the complex involving the
template strand, RNA polymerase and RNA molecule. In independent termination, a
loop forms at the end of the RNA molecule, causing it to detach itself. Termination in
eukaryotes is more complicated, involving the addition of additional adenine nucleotides
at the 3' of the RNA transcript (a process referred to as polyadenylation).
• Processing. After transcription the RNA molecule is processed in a number of ways:
introns are removed and the exons are spliced together to form a mature mRNA
• molecule consisting of a single protein-coding sequence. RNA synthesis involves the
normal base pairing rules, but the base thymine is replaced with the base uracil.

10. Translation
• In translation the mature mRNA molecule is used as a template to
assemble a series of amino acids to produce a polypeptide with a
specific amino acid sequence. The complex in the cytoplasm at which
this occurs is called a ribosome.
• Ribosomes are a mixture of ribosomal proteins and ribosomal RNA
(rRNA), and consist of a large subunit and a small subunit.

11. • Initiation. The small subunit of the ribosome binds at the 5' end of the
mRNA molecule and moves in a 3' direction until it meets a start codon
(AUG). It then forms a complex with the large unit of the ribosome complex
and an initiation tRNA molecule.
• Elongation. Subsequent codons on the mRNA molecule determine which
tRNA molecule linked to an amino acid binds to the mRNA. An enzyme
peptidyl transferase links the amino acids together using peptide bonds.
The process continues, producing a chain of amino acids as the ribosome
moves along the mRNA molecule.
• Termination. Translation in terminated when the ribosomal complex
reached one or more stop codons (UAA, UAG, UGA).

12. • The ribosomal complex in eukaryotes is larger and more complicated
than in prokaryotes. In addition, the processes of transcription and
translation are divided in eukaryotes between the nucleus
(transcription) and the cytoplasm (translation), which provides more
opportunities for the regulation of gene expression.

13. Gene regulation
• Gene regulation is a label for the cellular processes that control the
rate and manner of gene expression. A complex set of interactions
between genes, RNA molecules, proteins (including transcription
factors) and other components of the expression system determine
when and where specific genes are activated and the amount of
protein or RNA product produced.
• Some genes are expressed continuously, as they produce proteins
involved in basic metabolic functions; some genes are expressed as
part of the process of cell differentiation; and some genes are
expressed as a result of cell differentiation.

14. Mechanisms of gene regulation include:
• Regulating the rate of transcription. This is the most economical
method of regulation.
• Regulating the processing of RNA molecules, including alternative
splicing to produce more than one protein product from a single
gene.
• Regulating the stability of mRNA molecules.
• Regulating the rate of translation.

15. History and Evolution

16. • Traditional Methods:
Northern blot
quantitative reverse transcription PCR (qRTPCR)
serial analysis of gene expression(SAGE) and
DNA microarrays.

17. Northern blot
• Northern blot analysis is a low throughput method that uses
electrophoresis to separate RNA by size.
• The separated RNA is transferred onto a nylon membrane and
immobilised to the membrane through covalent linkage by UV light or
heat.
• A labelled short oligonucleotide sequence or probe that is
complementary to a sequence in the target transcript is introduced
onto the membrane and its hybridisation to the target transcript is
detected via the use of X-ray.

19. • The Northern blot procedure is useful for determining RNA size and
to detect alternative splice products.
• However, Northern blot uses RNA without conversion into
complementary DNA (cDNA), therefore, the quality of quantification
is compromised by even low levels of RNA degradation.
• Northern blot has also relatively low sensitivity, due to non-specific
hybridisation, requires the use of radioactivity and requires greater
amounts of RNA compared to qRTPCR.

20. quantitative Reverse Transcription PCR (qRTPCR)
• qRTPCR involves the reverse transcription of the target RNA into
cDNA followed by PCR of the cDNA to amplify the signal for detection.
• It is a fluorescence-based real-time reaction method that allows for
detection and relative quantitation of target RNAs.
• The qRTPCR method has improved to enable high throughput
multiplexed reactions to quantify multiple genes in a single reaction.
• However, the throughput capability of the current technology of
qRTPCR remains on the order of only hundreds of known transcripts
in one assay, and is not adept for transcriptome-wide gene expression
analysis.

21. Serial Analysis of Gene Expression(SAGE)
• It is gene expression profiling method that involves creating cDNA
which is biotin-labelled at the 3′-end of the cDNA. Short sequence
tags of 14 or 21 bp that can uniquely identify specific transcripts are
extracted using restriction enzymes (Neisseria lactamica III). Nla III
cleaves the cDNA at the 5′-end of every CATG site.
• The cleaved sequence closest to the 3′-end of the cDNA is isolated
using streptavidin beads. This isolated sequence is shortened again to
contain only the CATG and the next ten nucleotides.
• This final sequence is the SAGE tag. These tags are ligated and cloned
into a vector which are Sanger sequenced to identify the sequence
tags.

22. • This method allows direct measurement of transcript abundance and
comparison between multiple samples. SAGE does not require a prior
knowledge of the transcript sequence and has been used for
discovery of novel transcripts and alternative splice isoforms.
• Nonetheless, SAGE is a costly technique with a laborious cloning
procedure.

25. DNA microarrays/ biochip/DNA chip
• The DNA microarray technology has superseded single-gene
approaches, allowing the measurement of RNA expression levels of
thousands of known or putative transcripts simultaneously and was
further developed to characterize the gene expression profile of a
complete eukaryotic genome (Saccharomyces cerevisiae).
• DNA microarray technology has enabled comprehensive
characterization and/or comparison of expression signatures of
various cell types and disease phenotypes.
• Gene profiling with microarrays have provided evidence of molecular
heterogeneity in cancer.

26. • Despite the success of gene expression profiling using DNA
microarrays in its contribution to the field of biology, the technique
remains limited by its requirement of a prior knowledge of the genes
of interest.
• To overcome this limitation, tiling arrays have been developed. Prior
to the advent of massively parallel sequencing technology, tiling
arrays was the method of choice to identify novel transcribed regions.

27. Principle of DNA microarray
• The principle of DNA microarrays lies on the hybridization between the nucleic acid
strands.
• The property of complementary nucleic acid sequences is to specifically pair with each
other by forming hydrogen bonds between complementary nucleotide base pairs.
• For this, samples are labeled using fluorescent dyes.
• Complementary nucleic acid sequences between the sample and the probe attached on
the chip get paired via hydrogen bonds.
• The non-specific bonding sequences while remain unattached and washed out during the
washing step of the process.
• Fluorescently labeled target sequences that bind to a probe sequence generate a signal.
• The signal depends on the hybridization conditions (ex: temperature), washing after
hybridization etc while the total strength of the signal, depends upon the amount of
target sample present.

28. Requirements of DNA Microarray Technique
There are certain requirements for designing a DNA microarray system,
viz:
• DNA Chip
• Target sample (Fluorescently labelled)
• Fluorescent dyes
• Probes
• Scanner

31. Applications of DNA Microarray
• In humans, they can be used to determine how particular diseases affect
the pattern of gene expression (the expression profile) in various tissues, or
the identity (from the expression profile) of the infecting organism. Thus, in
clinical medicine alone, DNA microarrays have huge potential for diagnosis.
• Discovery of drugs
• Diagnostics and genetic engineering
• Proteomics
• Functional genomics
• Gene expression profiling
• Toxicological research (Toxicogenomics)

32. Advantages of DNA Microarray
• Provides data for thousands of genes in real time.
• Single experiment generates many results easily.
• Fast and easy to obtain results.
• Promising for discovering cures to diseases and cancer.
• Different parts of DNA can be used to study gene expression.

33. Disadvantages of DNA Microarray
• Expensive to create.
• The production of too many results at a time requires long time for
analysis, which is quite complex in nature.
• The DNA chips do not have very long shelf life.

34. Thank You..