BIOTECHNOLOGY PPT.pptx

GENOME
SEQUENCING
DNA SEQUENCING -
(Sanger method)

INTRODUCTION
 Study of the complete set of genetic instructions,or
genome,of an organism.
 Involves analyzing the structure,function, and evolution
of genomes,as well as the interactions between the
genes and the environment.
 The genome of an organism contains information for
making proteins, regulating gene expression,and
responding to the environment
 Genomics has become an increasingly important field
of study in recent years,as advances in technology
have made it possible to sequence entire genomes
more quickly and accurately than ever before.This has
led to a better understanding of the genetic basis of
many diseases,as well as new insights into evolution,
ecology,and agriculture

GENOMICS
Structural
genomics
Functional
genomics
Comparative
genomics
Epigenomics
Metagenomics

GENOMICS
 1.Structural genomics: This involves the study of the physical structure of
genomes, including the arrangement and organization of genes and non-
coding regions of DNA.
 2.Functional genomics: This involves the study of how genes and their
products (proteins) function within cells and organisms. This includes
understanding the roles of genes in cellular processes, as well as how
genes are regulated and expressed.
 3.Comparative genomics: This involves comparing the genomes of
different organisms to identify similarities and differences in their genetic
makeup. This can help us to understand evolutionary relationships
between species, as well as identify genes that are important for particular
functions
 .4.Epigenomics: This involves the study of changes in gene expression
that are not caused by changes in the DNA sequence itself, but rather by
chemical modifications to the DNA or associated proteins. These
modifications can have important effects on gene expression and can be
influenced by environmental factors.
 5.Metagenomics: This involves the study of genetic material from entire
communities of organisms, such as the microbiomes of humans or the soil.
This can provide insights into the diversity and function of these
communities, as well as potential applications in fields such as
biotechnology and environmental science.

TYPESOFGENOMES
 1. Nuclear genome: Genetic material= nucleus of eukaryotes.
 2. Mitochondrial genome: Genetic material found within the mitochondria of
eukaryotic cells. Smaller than the nuclear genome ; contains genes that are
important for mitochondrial function. ATPase 6, CYB, ND1, ND4
 3. Chloroplast genome: Plant organelles responsible for
photosynthesis=chloroplast. Like the mitochondrial genome, the chloroplast
genome is smaller than the nuclear genome contains genes important for
chloroplast – like genes of ribosomal and transport RNA. Independent replication
and transcription. Circular dna.
 4. Viral genome: Viral genomes can be either ss/dsDNA or ss/dsRNA, and can be
single-stranded or double-stranded. The viral genome contains all the genetic
information necessary for the virus to replicate and infect host cells.
 5. Bacterial genome:. Bacterial genomes can be composed of either DNA or RNA
and can be circular or linear. Bacterial genomes vary in size, with some bacteria
having very small genomes and others having large, complex genomes.
 6. Plasmid genome: Exogenously replicating, small, circular pieces of DNA that
can replicate independently of the chromosomal DNA, found in bacteria, and can
contain genes that confer antibiotic resistance/fertility/infecting properties

HOWAREGENOMICSANDGENETICSDIFFERENT
GENETICS
 Scope:Study of individual genes and their
inheritance patterns
 Scale: Focuses on the analysis of single genes
or small sets of genes
 Data analysis:Genetics involves the analysis
of small amounts of data, such as single
nucleotide polymorphisms (SNPs), while.
 Applications: Genetics is used for studying
inherited diseases,genetic disorders, and
genetic variation in populations, while
Techniques:Genetics relies on techniques
such as polymerase chain reaction (PCR), gel
electrophoresis,and gene sequencing.
GENOMICS
 study of the entire genome, including all
the genes, their interactions, and the
non-coding regions of DNA
 Deals with the analysis of the entire
genome and its organization.
 Involves the analysis of large amounts of
data, such as gene expression patterns,
DNA sequencing, and epigenetic
modifications
 Used for understanding complex
biological processes,such as
development, disease,and evolution
 Relies on high-throughput sequencing
technologies, microarrays,and
bioinformatics.

Whatissequencing?
Adenine Guanine Thymine Cytosine
DNA sequencing is the
process of determining
the nucleic acid
sequence
It includes any method
or technology that is
used to determine the
order of the four bases.

TECHNICALFOUNDATION
OFSEQUENCING
 Construction of cdna libraries-cDNA library
is a combination of cloned cDNA
(complementary DNA) fragments inserted into
a collection of host cells, which constitute
some portion of the transcriptome of the
organism and are stored as a "library“
 Dna hybridization-Two complementary
single-stranded DNA and/or RNA molecules
bond together to form a double-stranded
molecule.
 Restriction enzyme mapping- Map an
unknown segment of DNA by breaking it into
pieces and then identifying the locations of the
breakpoints. This method relies upon the use
of proteins called restriction enzymes, which
can cut, or digest, DNA molecules at short,
specific sequences called restriction sites
 PcR amplification- A laboratory technique
for rapidly producing (amplifying) millions to
billions of copies of a specific segment of
DNA, which can then be studied in greater
detail.

TYPESOFGENOMESEQUENCING
WHOLE GENOME
• In genome assembly,a
combination of short and
long pieces of DNA are
sequenced to cover as much
of the genome as possible
and minimise the risk of
there being any gaps in the
final sequence.
• METHODS- Whole Genome
Shotgun Sequencing
(WGS), NGS Accelerates
WGS,
TARGETED GENOME
• Targeted sequencing means
researchers can focus on
sequencing specific areas of
interest within the genome.
• One common use of targeted
sequencing is to look for single
nucleotide polymorphisms
(SNPs)
• SNPs are single base changes
in the DNA sequence.They are
the most common type
of genetic variation between
us, and can be used to help
scientists find genes associated
with disease.
• METHODS- Maxam Gilbert,
Sanger’s
EXOME PULLDOWN
• All of the exons in a genome,
which consist of the DNA that
contains the instructions to
make proteins= EXOME
• Exome sequencing is only
looking at a very small
section of the genome,it
enables scientists to focus on
the parts of the DNA
sequence that are more likely
to be linked to disease,the
genes.
• Quicker and cheaper than
sequencing the whole
genome.

Maxam-Gilbert/Chemical
Degradation
 Method of DNA sequencing
developed by Allan Maxam
and Walter Gilbert in 1976–
1977
 In the Maxam-Gilbert method,
DNA fragments radiolabeled at
their 5′ ends are chemically
cleaved at distinct nucleotides
(e.g.,A and G, G, C and T, or C).
The cleaved fragments are
then separated by gel
electrophoresis and detected
by autoradiography

Procedure
 Heat to form ssDNA
 Cool for the binding of primers
to the single stranded template
 PCR ( elongate as well as
terminates when ddNTPs are added)
 Collect the DNA strands
 Take 4 test tubes
 Add all required mixtures
 one type of ddNTPs -
A, T, C or G - is added to each
 Gel Electrophoresis

MECHANISMOFSANGER’SSEQUENCING
 In Sanger sequencing, a single-stranded DNA template is mixed with a
DNA primer, DNA polymerase, and four types of deoxynucleotides
(dNTPs) that serve as the building blocks for the complementary strand.
 In addition to the regular dNTPs, small amounts of fluorescently labeled
dideoxynucleotides (ddNTPs) are also included in the reaction mix.
 These ddNTPs are similar to dNTPs but lack a 3' hydroxyl group, which
prevents further DNA synthesis once they are incorporated into the
growing DNA chain.
 As DNA polymerase extends the new strand, it will sometimes incorporate
a ddNTP instead of a regular dNTP, leading to chain termination at a
specific base. The result is a series of partially completed DNA strands of
varying lengths, each ending in a fluorescently labeled ddNTP.
 The fragments are then separated by size using gel electrophoresis, with
shorter fragments migrating faster through the gel than longer ones.
 Finally, the fragments are detected using a fluorescence scanner, which
detects the fluorescent label on each fragment and generates a signal that
corresponds to its length.
 By analyzing the sequence of the fragments from shortest to longest, the
original sequence of the template DNA can be determined

Finaldetectionofsequenceandanalysis
 Gel is sandwiched against X-ray film
 Radioactive DNA emits beta particles that
expose the film
 The ends of the fragments will be labeled with
dyes that indicate their final nucleotide.
 Capillary gel electrophoresis
 The fragments will separate.
 There’s a finish line at the end of the tube, its
illuminated by a laser, allowing the attached dye
to be detected.

MARKERSUSEDINGENOMESEQUENCING
 Fluorescent dyes: These are used to label the chain-terminating nucleotides, allowing
them to be detected by fluorescence rather than by radioactivity. This makes the
sequencing process faster, more accurate, and less hazardous
 Capillary electrophoresis: This technique uses thin, glass capillaries to separate the
labeled fragments based on their size and charge. Capillary electrophoresis is faster and
more efficient than traditional gel electrophoresis, and it can be automated to handle
large-scale sequencing projects.
 High-throughput sequencing: This approach uses massively parallel sequencing to
generate large amounts of sequence data in a short amount of time. High-throughput
sequencing methods, such as Illumina sequencing, allow for the simultaneous
sequencing of millions of DNA fragments, making it ideal for large-scale genomic
studies.
 Next-generation sequencing: This is a broad term that encompasses several different
methods of high-throughput sequencing, including Illumina sequencing, 454
sequencing, and Ion Torrent sequencing. Next-generation sequencing methods are
characterized by their speed, accuracy, and ability to generate large amounts of
sequence data in a short amount of time.
 Pyrosequencing: This method uses a chemical reaction to generate a light signal in
response to the incorporation of nucleotides into a growing DNA strand. Pyrosequencing
is faster and more accurate than traditional Sanger sequencing, and it can be used to
detect small variations in DNA sequences, such as single nucleotide polymorphisms
(SNPs).

MILESTONES IN SEQUENCING
• 1990 : HGP launched
• 2000: Release of first full genomic sequence for the fruit fly, Drosophila chosen for sequencing owing to its
importance as a model organism existence.
• 2001: set out to identify the sequence, of all DNA bases to obtain the ‘genetic blueprint’ of humans. In 2001,
first draft of the human genome, obtained by shotgun sequencing,The second phase of the project, was
completed in 2003.
• 2004: Sequencing the unculturable majority, via Metagenomics—Reconstruction of microbial communities
from sequencing data — by providing approaches for unbiased, culture-independent analysis of DNA
directly from environmental samples using sequencing technologies
• 2007: The development of ChIP–seq, which combined chromatin immunoprecipitation with high-throughput
next-generation sequencing, enabled the genome-wide interrogation of chromatin binding patterns of
different proteins, lending insight into gene regulation mechanisms, development and epigenetics.
• 2008: sequencing revolution in cancer. Ley et al. presented the first whole-genome sequence of a
cytogenetically normal acute myeloid leukaemia sample, showing that cancer genome sequencing can
identify disease-associated mutations and druggable targets
• 2010: Neanderthal’s genome sequencing marked a turning point for the palaeogenomics field, making it
possible to assemble an ancient genome from next-generation sequencing.

APPLICATIONSOF
GENOMERESEARCH
 Pharmaceutical: Researchers have
identified specific genes that contribute to
certain types of cancer, which has led to
the development of targeted therapies.
 Agriculture: Genome research is helping
to develop crops that are more resistant to
pests and disease, which could increase
crop yields and reduce the need for
pesticides. Eg Rice and Wheat using
CRISPR CAS9 and soyabean using TALEN
 Environmental applications: Studying
the genomes of endangered species for
conservation; NGS (New Generation
Sequencing). Sister lineage between
Dryas monkey and Snub-nosed monkey
 Evolutionary studies: Sequencing the
genome of the Neanderthal has helped us
to better understand the evolutionary
history of modern humans.

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