description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it

1. genomics
D.INDRAJA

2. Gene:
It is a unit of heridity which is transferred
from a parent to offspring and held to determine
some characteristic to offspring
Genome:
The entire set of genetic information in an
organism
It is encoded in DNA or RNA in case of
many viruses
coined by HANS WINKLERin 1920
Genomics
field of study where entire genome is studied
 coined by THOMAS RODERICK

3. Goals of genomics
• Compile the genomic sequences of organisms
• Search out the location of the genes for analyzing
spatial relationships and annotate the gene set in a
genome
• Learn the function of genes and their influence
• Establish how gene expression profiles of a cell vary
under different conditions.
• Compare gene and protein profiles among different
organisms to learn about evolutionary relationships.

4. • The field of genomics comprises of two main areas: 1.Structural genomics
2. Functional genomics
Structural genomics
• Structural genomics helps to describe the 3- dimensional structure of every
protein encoded by a particular genome.
• The principal difference between structural genomics and traditional
structural prediction is that structural genomics attempts to determine the
structure of every protein encoded by the genome, rather than focusing on
one particular protein.
• This genome-based approach allows for a high- throughput method of
structure determination by a combination of experimental and modeling
approaches.
• experimental methods using genomic sequences
• modeling-based approaches .... based on sequence or structural homology of
a protein of known structure
• or based on chemical and physical principles for a protein with no homology
to any known structure.

5. Functional genomics
• Functional genomics is a field of molecular biology that attempts to
describe gene (and protein) functions and interactions. Functional genomics
make use of the vast data generated by genomic and transcriptomic projects
(such as genome sequencing projects and RNA sequencing).
• Functional genomics is the study of how the genome, transcripts (genes),
proteins and metabolites work together to to produce a particular phenotype.
• Together, transcriptomics, proteomics and metabolomics describe
the transcripts, proteins and metabolites of a biological system, and the
integration of these data is expected to provide a complete model of the
biological system

6. • Functional genomics focuses on the dynamic expression of gene products in
a specific context, for example, at a specific developmental stage or during a
disease.
Why we need to study?
• It is estimated that approximately 30% of the open reading frames in a fully
sequenced organism have unknown function at the biochemical level and are
unrelated to any known gene.
• This is why recently the interest of researchers has shifted from genome
mapping and sequencing to determination of genome function by using the
functional genomics approach.
• Example A single gene can give rise to multiple gene products. RNA can be
alternatively spliced or edited to form mature mRNA. Besides, proteins are
regulated by additional mechanisms such as posttranslational modifications,
compartmentalization and proteolysis. Finally, biological function is
determined by the complexity of these processes.
• The goal of functional genomics is to understand the relationship between
an organism’s genome and its phenotype.

7. There are several specific functional genomics approaches depending on what
we are focused on:
• DNA level (genomics and epigenomics);
• RNA level (transcriptomics);
• protein level (proteomics);
• metabolite level (metabolomics).
TECHNIQUES
• Functional genomics uses mostly multiplex techniques to measure the
abundance of many or all gene products such as mRNAs or proteins within
a biological sample.
• A more focused functional genomics approach might test the function of all
variants of one gene and quantify the effects of mutants by using sequencing
as a readout of activity.
• Together these measurement modalities try to quantitate the various
biological processes and improve our understanding of gene and protein
functions and interactions.

8. Techniques at DNA level
• Epistasis is a phenomenon in genetics in which the
effect of a gene mutation is dependent on the presence
or absence of mutations in one or more other genes
Genetic
interactions
• Techniques have been developed to identify sites of
DNA-protein interactions. These include Chip-
sequencing, CUT&RUN sequencing and Calling Cards.
DNA/protein
interactions
• These regions of open chromatin are candidate
regulatory regions(that are acessible). These assays
include ATAC-seq, DNase-Seq and FAIRE-Seq.
DNA accessibility
assays

9. Techniques at RNA level
• Microarrays
Microarrays measure the amount of mRNA
in a sample that corresponds to a given
gene or probe DNA sequence. Probe
sequences are immobilized on a solid
surface and allowed to hybridize with
fluorescently labelled “target” mRNA. The
intensity of fluorescence of a spot is
proportional to the amount of target
sequence that has hybridized to that spot,
and therefore to the abundance of that
mRNA sequence in the sample

10. DISADVANTAGES
• gene expression studies were done with hybridization-
based microarrays. Issues with microarrays include cross-
hybridization , poor quantification of lowly and highly expressed
genes, and needing to know the sequence a priori.
• Because of these technical issues, transcriptomics transitioned to
sequencing-based methods. These progressed from Sanger
sequencing of Expressed Sequence Tag libraries, to chemical
tag-based methods (e.g., serial analysis of gene expression), and
finally to the current technology, next-gen
sequencing of cDNA (notably RNA-Seq).

11. Serial analysis of
gene expression
Serial Analysis of Gene
Expression (SAGE) is
a transcriptomic technique used by
molecular biologists to produce a
snapshot of the messenger
RNA population in a sample of
interest in the form of small
tags(which are DNA by means of
cDNA) that correspond to fragments
of those transcripts and they are
analyzed

12. RNA-Seq
RNA-Seq (named as an abbreviation
of "RNA sequencing") is a particular
technology-
based sequencing technique which
uses next-generation
sequencing (NGS) to reveal the
presence and quantity of RNA in a
biological sample at a given moment,
analyzing the continuously changing
cellular transcriptome.

13. Massively Parallel
Reporter Assays
(MPRAs)
Massively parallel reporter assays is a
technology to test the cis-regulatory activity
of DNA sequences
STARR-seq
STARR-seq (short for self-transcribing
active regulatory region sequencing) is a
method to assay enhancer activity for
millions of candidates from arbitrary
sources of DNA. It is used to identify the
sequences that act as transcriptional
enhancers in a direct, quantitative, and
genome-wide manner
Perturb-seq combines
multiplexed CRISPR mediated gene
inactivations with single cell RNA
sequencing to assess comprehensive gene
expression phenotypes for each perturbation
Perturb-seq

14. Techniques at protein level
yeast two-hybrid
system
Two-hybrid screening (originally known
as yeast two-hybrid system or Y2H) is
a molecular biology technique used to
discover protein–protein
interactions (PPIs) and protein–DNA
interactions by testing for physical
interactions (such as binding) between
two proteins or a single protein and
a DNA molecule, respectively.

15. Affinity
chromatography
Affinity chromatography is a method of
separating biochemical mixture based on a
highly specific interaction
between antigen and antibody, enzyme an
d substrate, receptor and ligand,
or protein and nucleic acid. It is a type
of chromatographic laboratory
technique used for purifying biological
molecules within a mixture by exploiting
molecular properties, e.g. protein can be
eluted by ligand solution.

16. Mass spectrometry
Mass spectrometry (MS) is an analytical
technique that measures the mass-to-
charge ratio of ions. The results are
typically presented as a mass spectrum,
a plot of intensity as a function of the
mass-to-charge ratio. Mass spectrometry
is used in many different fields and is
applied to pure samples as well as
complex mixtures

17. Deep Mutational
Scanning
In Deep mutational scanning every
possible amino acid change in a
given protein is first synthesized.
The activity of each of these protein
variants is assayed in parallel using
barcodes for each variant. By
comparing the activity to the wild-
type protein, the effect of each
mutation is identified.
Loss-of-function
techniques
Mutagenesis, RNAi, CRISPR
screens

19. • In molecular biology there are number of techniques are
available to understand the function of the gene
• For identification of gene function there are two methods
used commonly
• Forward genetics (Classical genetics)
• Reverse genetics
Forward genetics
• A traditional approach to the study of gene function that begins with a
phenotype (a mutant organism) and proceeds to a gene that encodes the
phenotype
• It depends upon the identification and isolation of random mutation that
affect the phenotype of interest

20. Reverse genetics
• A molecular approach that begins with a genotype ( a DNA sequence) and
proceeds to the phenotype by altering the sequence or by inhibiting its
expression
• It is possible due to the advancement in the molecular genetics

21. Forward genetics vs reverse genetics

22. Techniques of forward genetics
• Initially scientist are depends on the mutation that are occurs naturally, but
after the discovery of mutagenic agent increases the rate of mutation
• First experimentally created mutation - using X rays - to induce X linked
mutation in Drosophila melanogaster- by H. J. Muller in 1927
• Two types of mutations were majorly used in the forward mutation –
A. Spontaneous mutation
B. Creating random mutation
Spontaneous Mutation
• It arises spontaneously from natural changes in DNA structure or from error
in the replication
• i.e. mutation results from both internal and external factors
• Where as the changes caused by the radiation or environmental chemicals
are called as induced mutation
Creating random mutation
• It depends upon the identification and isolation of random mutation that
affect the phenotype
• Radiation (X rays), chemical mutagen (EMS) and transposable elements
(insert within a coding region and disrupt the amino acid sequence ) are used
to create the mutation

23. NON RECOMBINANT RECOMBINANT
•Large-scale random
mutagenesis and screening
•Chemical mutagenesis or
TILLING
•Gene silencing(RNAi)
•Next generation sequencing.
•Homologous recombination
•Genome
edition(ZFNS,TALENS,CRISP
ER)
Reverse genetics techniques

24. Large-scale random mutagenesis and screening
• In such methods, cells or organisms are exposed to mutagens such
as UV radiation or mutagenic chemicals, and mutants with desired
characteristics are then selected.
• Hermann Muller discovered in 1927 that X-rays can cause genetic
mutations in fruit flies, and went on to use the mutants he created
for his studies in genetics.
• For Escherichia coli, mutants may be selected first by exposure to
UV radiation, then plated onto an agar medium. The colonies
formed are then replica-plated, one in a rich medium, another in a
minimal medium, and mutants that have specific nutritional
requirements can then be identified by their inability to grow in the
minimal medium.
• instead of screening for a particular phenotype, screen your gene of
interest for nucleotide changes
• Typically requires that screen 1000’s or 10,000’s of individuals
• This is done by performing PCR for gene of interest and looking
for slight differences in the migration of the PCR product on a gel
or column

25. Tilling
• Targeting Induced Local Lesions IN Genome (TILLING)
• Identifying point mutations in a specific gene by heteroduplex
analysis.
• Introduced in 2000, using the model plant Arabidopsis thaliana by
McCallum.
• Identified diverse versions of genes in a germplasm and acquiring
information about their gene function.
• An expansion of the TILLING technique is Eco TILLING, which can
be used to discover point mutations or polymorphisms in Natural
populations.
• Both can be used to identify unknown and known point mutations
from a set of candidate genes.

26. Steps involved
Seeds are mutagenized by EMS
M1 population is grown from mutagenized seeds
Resulting M1 plants are self fertilized to produce M2 generation
The M2 generation of individuals are used to prepare DNA
samples for mutational screening.
The DNA samples are pooled eight fold to maximize screening
efficiency

27. Arrayed on 96-well micro titer plates
Equal amounts of DNA from each individual sample.
The pooled DNA samples are amplified by PCR using primers,
Heteroduplex Formation by PCR amplification at the place of
mutation
heteroduplex is cleaved at the 3’ end of the mismatch by
Endonuclease Cel I.
The cleaved products can be separated easily by 10%

29. Gene silencing(RNAi)
• Long ds-RNA cleaved by an endo-
nuclease (Dicer) to form si-RNA.
• Once the si-RNA enters the cell it get
incorporated into other protein to form
RISC ( Argonaut , slicer)
• Si-RNA unwound to form single stranded
si-RNA.
• Si-RNA scan and find a complementary m-
RNA
• Bind to the targeted m-RNA and cleaved
it.

30. Next generation sequencing
•Technologies developed after that are known as next generation
sequencing.
•NGS enables the sequencing of biological codes at a very rapid pace
with low cost per operation.
•This is the primary advantage over conventional methods.
•For example Billions of short reads can be sequenced in one operation.

31. Homologous recombination
• Recombination is the exchange of genetic information between DNA
molecules; when the exchange is between homologous DNA molecules it is
called homologous recombination
• Works in bacteria, yeast, mice and other mammals
• Homologous recombination can be used to produce specific mutation in an
organism. Vector containing DNA sequence similar to the gene to be
modified is introduced to the cell, and by a process of recombination
replaces the target gene in the chromosome. This method can be used to
introduce a mutation or knock out a gene, for example as used in the
production of knockout mice