Lecture 6

TOOLS AND TECHNIQUES IN

BIOTECHNOLOGY
1. Tissue Culture
 Tissue Culture -The maintenance or growth of
tissue, in vitro, in a way that may allow
differentiation and preservation of their function.
 Totipotency-A cell characteristic in which the
potential for forming all the cell types in the adult
organism are retained.
Explant

 Cell, tissue or organ of

a plant that is used to
start in vitro cultures
 Many different explants
can be used for
micropropagation, but
axillary buds and
meristems are most
commonly used
Callus
Unorganized, growing mass of
cells
From Callus to Green Plants
2. Protoplast Fusion
 fusing together non reproductive
somatic cells
 Methods of Fusion
a. Polyethylene gylcol (PEG)
b. Electrofusion
Allotetraploid hybrids for seedless
triploid Citrus breeding
 Encore’ mandarin
 ‘Valencia’ sweet orange,
 ‘Encore’ Caffin’

Somatic hybrid plant in greenhouse.

Wu et al 2005, Euphytica (2005) 141: 229–235
Brassica napus
Thlaspi caerulescens
Zn and Cd removal-photo remediation

Protoplast fusion and plant phenotypes of B. napus+T. caerulescens somatic

hybrids and parental species. (Brewer et al 1999, TAG:99:761-771)
 3. PCR - Polymerase Chain Reaction
 A very quick, easy, automated method used
to make copies of a specific segment of
DNA
 widely used in molecular biology.
 derives its name from one of its key
components, a DNA polymerase used to
amplify (i.e., replicate) a piece of DNA by in
vitro enzymatic replication.
 As PCR progresses, the DNA thus
generated is itself used as template for
replication
The polymerase chain reaction (PCR)

 This sets in motion a chain reaction in which the

DNA template is exponentially amplified. With PCR
it is possible to amplify a single or few copies of a
piece of DNA across several orders of magnitude,
generating millions or more copies of the DNA
piece.

 PCR can be performed without restrictions on the

form of DNA, and it can be extensively modified to
perform a wide array of genetic manipulations.
 What’s needed….

1. DNA template
2. DNA primers that “bracket” the
desired sequence to be cloned
3. Heat-resistant DNA polymerase
4. DNA nucleotides
5. Thermocycler
Developed in 1983 by Kary Mullis, PCR is now a common and often
indispensable technique used in medical and biological research labs
for a variety of applications. These include DNA cloning for sequencing,
DNA-based phylogeny, or functional analysis of genes; the diagnosis of
hereditary diseases; the identification of genetic fingerprints (used in
forensics and paternity testing); and the detection and diagnosis of
infectious diseases. Mullis won the Nobel Prize for his work on PCR.
The polymerase chain
reaction (PCR)
PCR
• Repetitive amplification of a piece or region of DNA
• Numerous uses
– Straightforward amplification & cloning of DNA
– RT-PCR – reverse transcription coupled with PCR to
amplify mRNAs (cDNAs actually are template)
– Production of cDNA libraries
– Mutagenesis
– Sequencing
PCR Requirements
• DNA template
– DNA that will be amplified (copied)
• Oligodeoxynucleotide primers
– anneal to template to allow DNA replication
• thermostable DNA polymerase
– DNA polymerase extends the primers to synthesize
a copy of the template DNA
– thermostable polymerases allow automation and
repeated rounds of DNA denaturation
• deoxynucleotides and appropriate reaction conditions
– dNTPs are incorporated into synthesized DNA,
buffered pH, & Mg2+ to allow enzyme activity of
DNA polymerase
PCR: The Process
1. Begin with a DNA template
• Insert in vector
• 1st strand cDNA
• Genomic DNA

AAAAAAA
TTTTTTT
PCR: The Process
2. Denature template
3. Anneal primers

AAAAAAA
TTTTTTT
PCR: The Process

AAAAAAA

TTTTTTT
 PCR: The Process
Beginning of 2nd cycle
1&2
1. Melt newly synthesized DNA
from template
• New strands of DNA are
now also available as
templates
2. Anneal primers
3 3. Extend primers
 PCR: The Process

• Beginning of 3rd cycle

• Melt newly synthesized
DNA from template
– All new strands of
DNA are now also
available as
templates
• Anneal primers
• Extend primers
PCR: Yields
• How much amplification can be achieved?
– Each cycle of PCR theoretically doubles the
number of template molecules
– Therefore the rate of amplification is 2n
Where n is the number of amplification cycles

– This will reach a practical maximum yield due to

reagent (primer & dNTPs) concentration limits and
maximum rate due to limiting enzyme
concentrations. This upper limit is about 1x106 X
amplification.
 PCR: Yields
Example: Starting with 2ng of 5kb DNA template to amplify a 1Kb
insert, what is the theoretical yield after 20 cycles? After 30?
1. How many template molecules are there?
= 5000bp X 660g bp/mol bp = 3.3x106 g template/mol template

= 2x10-9 g template  3.3x106 g temp/mol temp = 6x10-16 mol

temp

= 6x10-16 mol temp X 6.02x1023 molec/mol = 3.64x108 molecules

2. How many molecules of insert can be made in 20 cycles? 30?

3.64x108 molecules x 220 = 3.8x1014 molecules – 106 X
3.64x108 molecules x 230 = 3.9x1017 molecules – 109 X
 Functions And Their
Associated Enzymes
Function Enzyme
• Melting DNA è Helicase
è SSB Proteins
è Topoisomerase
• Polymerizing DNA è DNA
Polymerase
• Providing primer è Primase
• Joining nicks è Ligase
 Components of a PCR Reaction

• Buffer (containing Mg++)

• Template DNA
• 2 Primers that flank the fragment
of DNA to be amplified
• dNTPs
• Taq DNA Polymerase (or another
thermally stable DNA polymerase)
 PCR 100 Melting Melting
30x

Temperature
94 oC Extension 94 oC
Annealing 72 oC
Primers
50
50 oC

0
T i m e 3’ 5’
3’ 5’

3’ 5’
5’ 5’
3’ 5’
5’

3’ 5’
5’ 3’ 5’
5’ 3’
5’ 5’
5’ 3’

5’ 3’
5’ 3’
PCR
Temperature 100
Melting
94 oC

50

0
T i m e

3’ 5’
5’ 3’
PCR

Temperature
100
Melting
94 oC

50

0
T i m e
3’ 5’

Heat

5’ 3’
PCR

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’ 3’
PCR 30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’

Heat
5’

5’

Heat
5’
5’ 3’
PCR 30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing oC
Primers 72
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’

5’
5’

5’
5’ 3’
 30x

Temperature
PCR 100
Melting
94 oC
Melting
94 oC
Annealing Extension
Primers 72 oC
50 50 oC

0
3’ 5’ 5’ T i m e
5’
5’
5’ 3’

Heat
5’

5’

Heat
5’
 30x

Temperature
PCR 100
Melting
94 oC
Melting
Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’

5’
5’

5’
5’
PCR 30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing oC
Primers 72
50 50 oC

0
3’ 5’ 5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’
Fragments of
defined length 5’
5’

5’
5’
DNA Between The Primers Doubles With Each Thermal Cycle

Number
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles
More Cycles = More DNA
Size Number of cycles
Marker 0 10 15 20 25 30
Theoretical Yield Of PCR
Theoretical yield = 2n x y
Where y = the starting
number of copies and
n = the number of thermal cycles
If you start with 100 copies, how many copies are
made in 30 cycles?
2n x y
= 230 x 100
= 1,073,741,824 x 100
= 107,374,182,400
How The Functions Of Replication Are Achieved
During PCR
Function PCR
• Melting DNA è Heat
• Polymerizing DNA è Taq DNA
Polymerase
• Providing primer è Primers are
added to the
reaction mix
• Joining nicks è N/A as fragments
are short
4. Gel electrophoresis

 Electrophoresis - the migration of charged

molecules in solution in response to an electric
field.
 rate of migration
1. the strength of the field;
2. the nett charge,
3. size and shape of the molecules
4. ionic strength,
5. viscosity
6. T0 of the medium in which the molecules are
moving.
a technique used for the separation of
DNA, RNA, or protein molecules
using an electric current applied to a
gel matrix.

performed in a gel composed of

agarose, polyacrylamide or starch.
Gel electrophoresis of macromolecules
Using restriction fragment patterns to distinguish DNA
from different alleles
5. DNA Fingerprinting

method of identification that

compares fragments of
deoxyribonucleic acid (DNA).
With the exception of identical twins,
the complete DNA of each individual
is unique.
DNA Fingerprint
An individual's unique sequence of
DNA base pairs, determined by
exposing a sample of the person's DNA
to molecular probes.
used as evidence in criminal law cases.
Also called genetic fingerprint.
A DNA fingerprint is constructed by:

1. Extracting a DNA sample from

body tissue or fluid such as hair,
blood, or saliva.
2. The sample is then segmented
using enzymes, and the segments are
arranged by size using a process
called electrophoresis.
3. The segments are marked with
probes and exposed on X-ray film,
where they form a characteristic
pattern of black bars—the DNA
fingerprint.

If the DNA fingerprints produced

from two different samples match,
the two samples probably came from
the same person.
 DNA fingerprints from a murder case

Whose blood is on the defendant’s clothing?

 6. MOLECULAR MARKERS
 any kind of molecule indicating the existence of a
chemical or physical process
 In medicine- can be a substance that is introduced in an
organism as a means to examine something. For example,
rubidium chloride is used as a radioactive isotope to
evaluate perfusion of heart muscle.
 In biology and medicine, a molecular marker (biomarker)
can be a substance native to the organism whose
detection indicates a particular disease state (for example,
the presence of an antibody may indicate an infection).
 In genetics, a molecular marker (identified as genetic
marker) is a fragment of DNA sequence that is associated
to a part of the genome
MOLECULAR MARKERS
 identifiable DNA sequences,
 found at specific locations on the
chromosomes,
 transmitted from one generation to the next.
 may be located in or near genes.
 Since DNA is the same in every cell, the
molecular markers can be identified by a DNA
test regardless of the developmental stage,
age, or environmental challenges experienced
by the organism.
 can be useful tools to both facilitate breeding
programmes (in Marker Assisted Selection -
MAS) or to aid in characterization of
collections of germplasm (or varieties)
 1. Variable number tandem repeat
(VNTRs)
 location in a genome where a short nucleotide
sequence is organized as a tandem repeat.
 can be found on many chromosomes, and often
show variations in length between individuals.
Each variant acts as an inherited allele, allowing
them to be used for personal or parental
identification.
 analysis is useful in genetics and biology
research, forensics, and DNA fingerprinting.
Schematicdiagram of a Variable Number of Tandem Repeats in 4 alleles.
Variations of VNTR (D1S80) allele lengths in 6 individuals.
Chromosomal locations of the 13 VNTR loci in the CODIS panel.
KINDS of MOLECULAR MARKERS:
 RFLPs
 RAPDs
 AFLPs
 microsatellites (SSR)
 SNPs.

Differences:
 technical requirements;
 the amount of time, money and labour needed
 the number of genetic markers that can be detected
throughout the genome
 the amount of genetic variation found at each marker
in a given population
RFLPs: Restriction Fragment Length
Polymorphisms
 markers detected by treating DNA with restriction
enzymes (enzymes that cut DNA at a specific
sequence).
 based on the differential hybridization of cloned
DNA to DNA fragments in a sample of restriction
enzyme digested DNAs;
 specific to a single clone/restriction enzyme
combination
 first molecular marker to be widely used.
 time-consuming and expensive and simpler
marker systems have subsequently been
developed
Polymorphisms in the lengths of particular restriction
fragments can be used as molecular markers on the
genetic chromosome
Randomly Amplified Polymorphic DNA (RAPDs)

 a molecular marker based on the

differential PCR amplification of a
sample of DNAs from short
oligonucleotide sequences
 based on the PCR amplification of random
locations in the genome of the plant.

 with this technique, a single oligonucleotide

is used to prime the amplification of
genomic DNA.
Randomly Amplified Polymorphic DNA (RAPDs)
 RAPD markers are decamer (10
. nucleotides length)
DNA fragments from PCR
amplification of random segments of
genomic DNA with single primer of
arbitrary nucleotide sequence
 able to differentiate between
genetically distinct individuals,
although not necessarily in a
reproducible way.
 does not require any specific
knowledge of the DNA sequence of
the target organism:

the identical 10-mer primers will or

will not amplify a segment of DNA,
depending on positions that are
complementary to the primers'
sequence.
Microsatellites -simple sequence tandem
repeats (SSTRs).

 Microsatellites, or Simple Sequence

Repeats (SSRs), are polymorphic loci
present in nuclear and organellar DNA that
consist of repeating units of 1-6 base pairs
in length.
 They are typically neutral, co-dominant and
are used as molecular markers which have
wide-ranging applications in the field of
genetics, including kinship and population
studies.
 Repeat units are generally di-, tri- tetra- or
pentanucleotides.

 Ex: a common repeat motif in birds is ACn, where the

two nucleotides A and C are repeated in bead-like
fashion a variable number of times (n could range from
8 to 50).

 They tend to occur in non-coding regions of the DNA

(this should be fairly obvious for long dinucleotide
repeats) although a few human genetic disorders are
caused by (trinucleotide) microsatellite regions in
coding regions.

 On each side of the repeat unit are flanking regions that

consist of "unordered" DNA.

 The flanking regions are critical because they allow

development of locus-specific markers
 can also be used to study gene
dosage (looking for duplications or
deletions of a particular genetic region
Advantages of microsatellites as
genetic markers
 Locus-specific (in contrast to multi-locus markers
such as minisatellites or RAPDs)
 Codominant (heterozygotes can be distinguished
from homozygotes, in contrast to RAPDs and AFLPs
which are "binary 0/1")
 PCR-based (need only tiny amounts of tissue;
works on highly degraded or "ancient" DNA)
Highly polymorphic ("hypervariable") -- provides
considerable pattern
 Useful at a range of scales from individual ID to
fine-scale phylogenies
Comparison of marker systems (Korzun 2003) based on rice

Feature RFLPs RAPDs AFLPs Microsat SNPs

s
Amount of DNA required (μg) 10 0.02 0.5-1.0 0.05 0.05
Quality of DNA required high high moderate moderate high
PCR-based no yes yes yes yes
Number of polymorphic loci 1.0-3.0 1.5-50 20-100 1.0-3.0 1.0
analyzed per analysis
Ease of use not easy easy easy easy easy
Amenable to automation low moderate moderate high high

Reproducibility high unreliable high high high

Development cost low low moderate high high

Cost per analysis high low moderate low low
 7. Microarray, or ‘chip’,
technology
 Microarrays are arrangements of small
spotsof DNA fixed to glass slides or nylon
membranes
 The technology allows monitoring of the
whole genome at once
 The underlying principle of chips is base-
pairing or hybridisation between short
probes and complementary DNA sequences
 Microarrays are constructed using cDNAs
(cDNA arrays), genomic sequences or
oligonucleotides synthesised in silico (‘DNA
chips’)
DNA microarray assay for gene expression
DNA microarray assay for gene expression
8. DNA SEQUENCING

 method for determining the order of

the nucleotide bases,
adenine, guanine, cytosine, and
thymine, in a molecule of DNA.
 Knowledge of DNA sequences of
genes and other parts of the genome
of organisms has become
indispensable for basic research
studying biological processes, as well
as in applied fields such as diagnostic
or forensic research.
DNA sequencing methods:

1. Maxam-Gilbert

2. Sanger (dideoxy sequencing or chain

termination)
Sequencing of DNA by the Sanger method (Layer 1)
Sequencing of DNA by the Sanger method (Layer 2)
DNA Sequencing by the Sanger method (Layer 3)
Sequencing of DNA by the Sanger method (Layer 4)
DNA sequencing: advantages
and disadvantages
Advantages:
 Results
are highly reproducible
 Maximum amount of information content

Disadvantages:
 Costs are still high
 Technically demanding
Applications

Evolutionary studies
 Calculations of genetic variation
 Comparative genomics
 Creating PCR assays (making primers to
convert any marker to a PCR-based
marker)
Alternative strategies for sequencing an entire genome
Genome Sizes and Numbers of Genes
9. rDNA Technology
(GENETIC ENGINEERING)

The transfer of genetic information

(DNA) from one organism to another
The best known application of
molecular genetics
 Genetic engineering,
recombinant DNA technology,
genetic
modification/manipulation (GM)
and gene splicing
 terms that apply to the direct
manipulation of an organism's genes.
 Genetic engineering uses the
techniques of molecular cloning and
transformation to alter the structure
and characteristics of genes directly.
 Cloning (in biology)
 the process of producing populations of
genetically-identical individuals that occurs
in nature
 organisms such as bacteria, insects or
plants reproduce asexually.
 Cloning ( biotechnology)-processes used to
create copies of DNA fragments (molecular
cloning), cells (cell cloning), or organisms.
 generally, the term refers to the production
of multiple copies of a product such as
digital media or software.
Cloning of any DNA fragment
essentially involves four steps
1.fragmentation - breaking apart a
strand of DNA
2.ligation - gluing together pieces of
DNA in a desired sequence
3.transfection - inserting the newly
formed pieces of DNA into cells
4. screening/selection - selecting out
the cells that were successfully
transfected with the new DNA
ELEMENTS OF MOLECULAR CLONING
 Method of breaking and joining DNA molecules
derived from different sources-recombinant DNA
technique.
 Suitable gene carrier that can replicate both itself and
foreign DNA segment linked to it.
 Means of introducing the composite DNA molecules
or chimera into a functional host cell and
 A method of selecting from a large population of cells
and clone of recipient cells that has required a
molecular chimera.
Restriction Enzyme
 RE

RE

Plasmid DNA
(Vector)
DNA from donor organism
 Foreign DNA
pSC 101 plasmid

replicator Cleavage Cleavage 1

2 3
Sites Sites

Cleavage by
Tetracyline AA T T AA T T AA T T
Endonucleus
Resistance
T TAA T TAA T TAA
Annealing

DNA Ligase

Plasmid Chimera

Transformation

Plasmid

Replication

Daughter Cells
Basic Techniques in rDNA
technology
 Manipulation of DNA in vitro
 Transfer of the construct into a host
cell (transformation ) and maintenance
of the cloned DNA in the cell
 Cloning or production of many
identical progeny, of phages or
plasmids
 Identification and selection of the
transformed host cells (those which
have taken up the constructs)
 Manipulation of the DNA construct to
ensure production of the protein
encoded by the cloned sequence
90
DNA Cloning in Bacteria
DNA Cloning in Bacteria
Agrobacterium tumefaciens
Agrobacterium in nature
History of Agrobacterium research
 1907 Agrobacterium was identified as the
causative agent of crown gall disease
 1940 Uncontrolled cell proliferation did not
require the continuous presence of
Agrobacterium
 1960 Tumor cells produced opines (modified
amino acids)
 1970 Discovery of tumor inducing plasmid or
Ti plasmid in Agrobacterium
 Using the Ti plasmid as a vector for genetic engineering in plants

Potential Applications
Genetically modify plants to...
– produce vaccines in their fruit (e.g. polio vaccine)
– be resistant to disease and pests
– require less fertilizer, pesticides and herbicides
– have a higher nutritional value
Overview of Agrobactrium mediated transfer
Plant Transformation and
Production of Transgenic Plants
Agrobacterium-mediated transformation of
indica rice cultivar IR64:

Kumar et al 2005
Plant Molecular Biology
Reporter 23: 67–73,

A) 1-month-old mature seed-derived calli, (B) proliferating calli at the end of second round of selection on
hygromycin (50 mg/L), (C) proliferating calli during third round of selection on hygromycin (50 mg/L), (D)
shoot regeneration on media containing hygromycin (30 mg/L).
Engineering of ‘Golden rice’
two genes (one
bacterial gene, two
daffodil genes
enabling the
endosperm to express
a carotenoid
biosynthetic pathway
and produce β- Datta el al 2003
carotene (provitamin
A) (Ye et al., 2000).

Co-transformation of multiple genes for carotenoid biosynthesis to produce

‘Golden rice’. Polished grains from transgenic indica rice cultivar IR64 showing
the white endosperm of wild-type plants (left) and yellow endosperm of
transgenic plants (right). Reproduced from Datta et al. (2003)
Gene Gun Method
Gene Gun Method
Plant Transformation and
Production of Transgenic Plants
The host cells that take The protein product encoded
up the recombinant by the cloned gene or the
DNA are called insert is produced by the host
transformed cells or cell.
recombinant bacteria
or a GM bacteria.
 + Bacteria carrying
Ti plasmid with
gene of interest
Plant tissue
culture

+ =
BIOLISTIC

GMO
 Yieldgard MON 810 Transgenic cassette

Roundup Ready GA 21 Transgenic cassette

Roundup Ready NK 603 Transgenic cassette

 Bt
Protein
toxic corn plant
mRNA becomes
to corn
resistant
borer to corn
is borer
produced
by corn
Bt gene in
corn plant cells
 1952, the transfer of nucleus from
frog blastocyst into enucleated oocyte
 In 1975, protocol to transfer the
nucleus of a fully differentiated Xenopus
cell into enucleated cell and produced
tadpoles
 In 1989, cloned mammals by taking
nuclei from the blastocyst of sheep
embryo,
 In 1996, nuclei were taken from
cultured embryonic sheep cells
 1997 Wilmut et al got the nuclei from
cultured breast epithelial cells or a fully
differentiated cell, producing the most
famous sheep in the world, Dolly
Dolly : The life and death of the cloned sheep
1. Fused cells (nuclei from mammary gland)
were cultured in oviducts of sheep.
2. After 6 days, 29 of 277 developed into
morula
3. About 1-3 embryos were transferred to 13
surrogate female parents and only one
became pregnant.
4. After 148 days pregnancy, on July 5, 1996
Dolly was born at 6.6 kg and was named
after the singer Dolly Parton.
5. Dolly grew into an adult sheep and bore
her own offspring.
6. Average life expectancy of sheep is 12 years but in
2002 (at 5 years old) Dolly was diagnosed with
arthritis which was unusual at that age.
7. February 14, 2003 she was put to sleep after being
diagnosed with progressive lung disease.
11/23/2020 115
11/23/2020 116
 Microinjection of DNA or a nucleus into Cell
Potential Applications
1. Germ line Gene Therapy—inject therapeutic gene into an egg cell (affects
future generations)
2. Somatic Gene Therapy—Inject therapeutic gene into a somatic cell, culture &
reinsert into an individual
3. Cloning—inject nucleus into an enucleated egg, culture & implant into a surrogate
mother.

Drawback: Inefficient means of gene transfer

Use of a Retrovirus
for Gene Therapy

Applications
Somatic Gene Therapy to
treat
• Gaucher Disease
• SCID’s “Bubble Boy”
(Severe Combined Immune
Difficiency)
 Transgenic “Pharm” animals

Potential Applications
• Genetically modify
mammals to produce
therapeutic peptide drugs
(e.g. insulin, )
• Isolate and purify drug
from the milk
• Potentially a more cost
effective method to
produce pharmaceuticals
Cloning Humans?????????? ??

11/23/2020 121
10. Bioinformatics:
Shaping the Future of
Biotechnology
DNA: The Information Storage
Molecule
DNA

trait

transcription translation
On DNA, RNA, Proteins
DNA - Made up of the nucleic
acids adenine, guanine, cytosine,
thymine (A, G, C, T)

RNA - Made up of the nucleic

acids adenine, guanine, cytosine,
uracil (A, G, C, U)

Proteins – Made up of amino

acids. Make up enzymes,
antibodies, and most cellular
components
Advances in Molecular Biology
Current Genome Listing Available total genomes: 243
Available total peptides: 915554

Some examples: Domain Species name

Bacteria Haemophilus influenza, KW20
Mycoplasma pneumoniae, M129
Escherichia coli, MG1655
Mycobacterium tuberculosis, H37RV
Vibrio cholerae, El Tor N16961
Archaea Methanococcus jannaschii, DSM 2661
Pyrococcus horikoshii, OT3
Eukaryota Saccharomyces cerevisiae, S288C
Caenorhabditis elegans
Drosophila melanogaster
Arabidopsis thaliana
Mus musculus
Homo sapiens
The Human Genome Project
The Present Challenge

 Handling a huge volume of data

- if compiled in books, the data would run into 200
volumes of 1000 pages each
- reading alone would require 26 years working
around the clock
 Bioinformatics?
The use of:

Statistics
Computer Programs and Programming
Mathematics

to store, organize, and index the

exponentially increasing
sequence information
Bioinformatics Tools

 Databases
– With DNA, RNA, or protein sequences
– With protein profiles
– With protein structures
 Computer Programs
– To study DNA, RNA, or protein
sequences
– To generate structures/models from
sequences
 Major Databases
 GenBank
- an annotated collection of all publicly available DNA
sequences developed by the US NIH (National Institute of
Health) / NCBI (National Center for Biotechnology
Information)

 The EMBL Nucleotide Sequence

Database
- composed of DNA and RNA sequences, genome
sequencing projects, and patent applications; Europe’s
primary nucleotide sequence database

 DNA Data Bank of Japan (DDBJ)

- collects nucleotide sequences from Japanese researchers,
as well as researchers from other countries; works in
collaboration with EMBL and NCBI/GenBank
 Major Databases
 Entrez
- the integrated text-based search and retrieval system that
serves as the gateway to six major searchable databases of
nucleotides, proteins, 3-D structures, genomes, taxonomy,
and literature

 The Genome Database

- contains all of the genomic mapping data from the human
genome project

 Online Mendelian Inheritance in Man

(OMIM) Database
- catalog of human genes and genetic disorders

 BLAST
- searches all available sequence databases for sequence
similarity with both nucleotide and protein sequences
What is BLAST?

Basic Local Alignment Search Tool

Developed by Altschul, Gish, Miller,
Myers, and Lipman in 1990
Used to search for related sequences
available in the database
Used for comparing two or more
sequences for similarities
Go to http://ncbi.nlm.nih.gov/BLAST/
The BLAST Search Engine

Paste your DNA sequences

here
 Results of a
BLAST Search
Graphical representation of
alignments

Scores for each result

(includes name, annotation,
and BLAST scores

Alignment of the query

sequence against the
results sequence
Results of a BLAST Search

Scores for each result (includes name, annotation, and

BLAST scores
Results of a BLAST Search

Alignment of the query sequence against the

matching sequence
Applications in Biotechnology
Research

» Agriculture
» Human Health
» Industry
» Environment
» Wildlife Conservation
» Forensics
Bioinformatics in Agriculture
Production of GOLDEN RICE
Gene for beta- Organisms from Transform
carotene BLAST search RICE with
the genes
production
Available in
Daffodils
other species
but not the Erwinia
uredovora
rice plant

Clone gene from

DAFFODILS and a
MICROORGANISM
(Erwinia uredovora)
Bioinformatics in Agriculture

Gene for Blue Organism from

BLAST search
pigmentation Transform
ROSE with
Available in the gene
other species
but not the
ROSE
Clone gene from
PETUNIA
Bioinformatics and Human Health

 Genome databases may be used

to map a gene mutation leading
to a genetic disorder
 Genome databases are useful in
identifying and studying a human
pathogen
 Protein and Genome databases
may be used to create vaccines
and drugs
Bioinformatics and Human Health
Mapping a
genetic disorder Determine gene
function to:
Human Complete
DNA 1. Map the
genome location of
from 5 sequence
races diseases
2. Make cures
for human
diseases
3. Gene therapy
Bioinformatics and Human Health
Production of antibodies
Sequence Use
Known Use software
complete antigenic
pathogen to identify
genome, determinan
without a antigenic
translate ts to create
vaccine determinants
known vaccine
proteins
Bioinformatics and Human Health
Molecular detection of human pathogens
1. Microscopy, to
check what the
pathogen looks Sequence
like key
2. Cell culture, to genes BLAST
Isolate an get data on culture
unknown to identify
morphology =
the
pathogen identity of microbe
pathogen
from
patient 3. Blood Bioinformatics can help
Chemistry doctors make a correct
4. Isolation of diagnosis – and thus
DNA from find the proper cure
pathogen immediately
Ribosomal RNA-targeted
Identification
plasmid

16S rRNA gene

PCR
SEQUENCE

HOMOLOGY
SEARCH

IDENTIFICATION
Molecular Detection of
Bacteria and Viruses
SHOULD IDENTIFY A PROTEIN, OR A REGION OF A
PROTEIN THAT IS UNIQUE TO A BACTERIAL
SPECIES OR A VIRUS
Targ
Target
protein
et
in the regi
cytosol on
, of
membran the
e, or ira
append
ages
l
coat
prot
Molecular Detection of Bacteria
MAY IDENTIFY A REGION OF THE GENE OR
DNA WITH SEQUENCES UNIQUE OR SPECIFIC
TO THE SPECIES
3’TGCCGCTATACAACTGGGCGGTGGGTGGCGCTGCTG
GCGA
5’ACGGCGATATGTTGACCCGCCACCCACCGCGACGAC
CGCT

AAATCAATACATCGCGCTAACAGGCGTTGGAAAGCAA
GTCTC
TTTAGTTATGTAGCGCGATTGTCCGCAACCTTTCHTTC
AGAG

TTCTTACCTTGCTTATATGCAGCTGGCGAAAAACTACA
ACCC
BACTERIUM AAGAATGGAACGAAGAGACGTGCACCGCTTTTTGATG
TTGGG
 Molecular Detection of Bacteria

5’GAACGGCGATATGTTGACCC
PCR 3’CTTGCCGCTATACAACTGGGCGGTGGGTGGCGCTGCTGGC
primers 5’GAACGGCGATATGTTGACCCGCCACCCACCGCGACGACCG

AAATCAATACATCGCGCTAACAGGCGTTGGAAAGCAAGTCTC
TTTAGTTATGTAGCGCGATTGTCCGCAACCTTTCHTTCAGAG

TTCTTACCTTGCTTATATGCAGCTGGCGAAAAACTACAACCC
AAGAATGGAACGAAGAGACGTGCACCGCTTTTTGATGTTGGG

TGCCAATACCCTGTTCACCCTTGAGTTTGGATTAAATGA’5
ACGGTTATGGGACAAGTGGGAACTCAAACCTAATTTACT’3
TTGAGTTTGGATTAAATGA’5

BACTERIUM Design PCR primers targeting the unique gene

sequences
 PCR PRIMERS

Table 1. PCR primers specific for the detection of bacterial pathogens

Bacteria Primer set Protein encode Expected size

Escherichia coli lamB outer coat protein 179 bp

Salmonella typhi hns DNA-binding protein 152 bp
Shigella dysenteriae ipaH invasion antigen 700 bp
Vibrio cholerae ctx AB cytotoxin AB 777 bp
PCR PROFILE ANALYSIS

Lanes: M, 100 bp ladder;

1-4, positive controls from
Escherichia coli, Salmonella typhi, Shigella
dysenteriae and Vibrio cholerae;
5 and 6, multiplex PCR using all 4 primers.
Bioinformatics and the Environment

 Search databases for genes from

microbes whose products may help
clean up the environment (degrade
heavy metals, petroleum oil, organic
and inorganic wastes)
 Use software to identify
microorganisms thriving in polluted
areas, which are environmental
pollutants
 Bioinformatics and the Environment

Identify
16S rRNA organism
gene through
sequence BLAST
analysis search
GUIMARAS Gene- Amplify
targeted gene for
PCR pollutant
degrading
enzyme
 Bioinformatics in the Industry
Bioinformatics may be used to search
for genes whose products can
improve organisms used in industry
such as
§ Microorganisms in food processing
industry
§ Microbial enzyme production used in
pharmaceutical, detergent, leather,
textile industry, etc
Bioinformatics in Forensics
• Bioinformatics is used in
forensics to:
§ Analyze the thousands of bands in
DNA fingerprinting
§ Analyze short DNA sequences of
individuals
§ Conduct statistical analysis of results
§ Search databases for suspects
Bioinformatics in Forensics
DNA Fingerprinting
Get DNA from tissue sample
(blood, skin, hair, sperm, swab)

Isolate DNA

Target regions of high polymorphism (variation)

DNA FINGERPRINT
 Bioinformatics in Forensics

DNA from Suspect

Use software identified
Suspects Fingerprint to analyze
with many banding OR
Child/
bands patterns
Mother/ Parentage
(>100)
Father clarified
 Bioinformatics
• Can help keep – or make – humans healthier
• Can help design a drug with the desired properties
and assess its therapeutic effects, theoretically
• Can help biotechnology produce better crops and
food products
• Can help clean up the environment
• Can help save endangered species and establish
definitive systems of classifications for the known
organisms
• Can help the law
• Is JUST A CLICK AWAY FOR RESEARCH
Thank You for listening!
Genetic modification

“pellet”

gene
insertion
DNA

DNA

nucleus plant chromosome

Genetic modification

Transformed plant cell

with gene of interest

Genetically modified plant

 Genetic engineering allows the transfer of
a specific gene for a desirable trait
Gene transfer to
plant tissue
Gene
infection
Agrobacterium

Vector Gene shooting

construct
Particle
Cloning sites bombardment
Gold bullets
Marker
gene Selection

Greenhouse
Variety selection/testing
Regeneration
Field testing
Biotechnology is a Natural Extension
of Traditional Plant Breeding
Bt corn MON810 – first GM crop
commercialized in the Philippines
Bt gene
Gene gun
Bt
gene

Soil microbe