(Chapter III) RDT Techniques and Apps

Previous Topics Discussed:
1. Central Dogma of Biology

2. Recombinant DNA Technology
RDT Techniques and Applications by Sarah May M. Querubin
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November 19, 2013
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RDT Steps:
1. Production of RDNA
Identification of gene of interest, DNA
donors, and vectors
Insertion of DNA into a cloning vector
2. Cloning/Amplification of Recombinant
Molecule
3. Screening
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Amplification & Screening

Amplification of the Recombinant
Vector
-for bacteria: the recombinant bacteria are
plated out on a nutrient medium so that the
recombinant DNA vector can be replicated, or
amplified.
-for bacteriophage vectors: Phage particles
containing recombinant DNA are mixed with
bacterial cells, and the mixture is then placed
on a culture medium under conditions that
produce a continuous lawn of bacteria
across the plate.
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Amplification & Screening

Screening
- selectable markers are
used
ex. pUC19 (puck-19)
plasmid cloning vector, has
an ampicillin-resistant gene
(ampR)
- color test is employed
(X-Gal)
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Movie Clip Available at

http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cf
m?coSiteNavigation_allTopic=1
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November 19, 2013
Isolation of gene
Genetic/Genomic Library
- a collection of
recombinant vector
clones; each clone
carrying a particular
segment coming from the
initial genome
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November 19, 2013
Isolation of gene
cDNA Libraries
- A collection of
DNA molecules
reverse transcribed
from the mRNAs in
a particular celltype
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cDNA Preparation
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November 19, 2013
YAC vector - about 10,000 bp

Inserted foreign DNA - 300,000
to 1.5 million bp
*at least 50,000 bp to be reliably
replicated and segregated
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Bacterial artificial chromosome (BAC)

>a derivative of the F factor plasmid that some bacteria
employ for transferring DNA between cells during bacterial
conjugation
>BAC vectors are modified forms of the F factor plasmid that can
hold up to 350,000 bp of foreign DNA and have all the components
required for a bacterial cloning vector, such as replication origins,
antibiotic resistance genes, and insertion sites for foreign DNA.
>One type of BAC facilitates the process of screening for

recombinant clones by including the SacB gene, which converts
sucrose (table sugar) into a substance that is toxic to bacteria. A
BamHI cloning site is located within the SacB gene, so when foreign
DNA is inserted into the BAC vector at this site, the SacB gene is
disrupted. When such a BAC vector is introduced into bacterial cells
grown in the presence of sucrose, only cells containing BAC vector
molecules with a foreign DNA insert will be able to grow. Those cells
receiving BAC vector with no DNA insert will fail to grow because the
SacB gene remains intact and produces a toxic substance from
sucrose.
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November 19, 2013
Screening DNA Libraries

-to facilitate easier step in obtaining
DNA materials needed for genetic
engineering
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November 19, 2013
Colony Hybridization Technique

Nucleic Acid Probe
-a single-stranded molecule of DNA
or RNA that can identify a desired
DNA sequence by base-pairing with
it
-Nucleic acid probes are labeled
either with radioactivity or with some
other chemical group that allows the
probe to be easily visualized
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DNA Fingerprinting by: Southern Blotting

RFLPs
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PAGE
Polyacrylamide
Gel Electrophoresis
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SDS-PAGE
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November 19, 2013
Polymerase Chain Reaction

(PCR)
Applications of PCR:
Amplifying DNA for cloning for
analysis
Testing for specific DNA sequences
of viruses
Comparing DNA molecules
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November 19, 2013
Sample computation for PCR preparation

C1
V1
Vf
X26 tubes
PCR Buffer
10x
2.5L
1x
65
MgCl2
25mM
2.5L
50 x 10 -6
65
dNTP
10mM
2.5L
25 x 10-6
65
i-TAQ
5 units/L
0.2L
1 unit
5.2
ddH2O
15.3L
397.8
Primer
1.0L
26
Template
Total
1.0L
25L
26
650L
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November 19, 2013
Suggested Cycling Parameters (iNtRON Biotechnology, Inc.)
PCR Cycle
Temp.
Initial denaturation
30-40
cycles
PCR product size

100-500bp
500-1000bp
1kb-5kb
94C
2min
2min
2min
Denaturation
94C
20sec
20sec
20sec
Annealing
50-65C
10sec
10sec
20sec
Extension
65-72C
20-30sec
40-50sec
1min/kb
Final Extension
72C
Optional. Normally, 2-5min
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November 19, 2013
http://www.nature.com/scitable/content/ne
0000/ne0000/ne0000/ne0000/5471131/PC
R_machine_2.jpg
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November 19, 2013
OTHER TECHNIQUES
AND APPLICATIONS
OF DNA TECHNOLOGY
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November 19, 2013
Monoclonal Antibodies
- uses immune-system cells that make
proteins called antibodies
- the specificity of antibodies makes a
powerful diagnostic tool
- Uses:
1. locate environmental pollutants
2. detect harmful microorganisms in food
3. distinguish cancer cells from normal cells
4. diagnose infectious diseases in humans,
animals and plants more quickly and more
accurately
5. protein purification
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MAbs + toxin = therapeutic compounds
Hybridomas
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Examples of MAbs
MAbs for Immune -Related Conditions
Muromomab-CD3 (OKT3) is used to prevent acute rejection of
organ transplants.
MAbs Used to Kill or Inhibit Cancer Cells
Rituximab (Rituxan) binds to the CD20 molecule that is found
on most B-cells and is used to treat B-cell lymphomas.
Angiogenesis Inhibitor
Bevacizumab (Avastin) blocks the vascular endothelial
growth factor (VEGF) receptor and has been approved for the
treatment of colorectal cancer.
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November 19, 2013
Cell Culture
Cell culture technology is the growing of cells
outside of living organisms under controlled
conditions (prokaryotic/eukaryotic)
1. Plant cell culture creation of transgenic
crops
2. Insect cell culture use of biological control
agents
3. Mammalian cell culture livestock
breeding, human in vitro fertilization
process, production technology for vaccines,
stem cell culture
Subculturing & Cell line
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November 19, 2013
http://www.foodprocessingtechnology.com/contractor_im
ages/biomerieux-australia/3culture-media.jpg
>Choice of media depends on the type of cell being cultured

>Asceptic Techniques
http://www.rnd
systems.com/r
esources/imag
es/6227.jpg
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November 19, 2013
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Applications of Cell Culture

1. Model systems for
Studying basic cell biology, interactions between diseasecausing agents and cells, effects of drugs on cells, process
and triggering of aging & nutritional studies
2.Toxicity testing
Study the effects of new drugs
3. Cancer research
Study the function of various chemicals, virus & radiation to
convert normal cultured cells to cancerous cells
4. Virology
Cultivation of virus for vaccine production, also used to study
there infectious cycle.
5. Genetic Engineering
Production of commercial proteins, large scale-production
of viruses for use in vaccine production e.g. polio, rabies,
chicken pox, hepatitis B & measles
6. Gene therapy
Cells having a functional gene can be replaced to cells which
are having non-functional gene
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Cloning
- Allows scientists to generate a population of
genetically identical molecules, cells, plants or
animals.
Molecular or Gene Cloning

Animal Cloning
- Artificial Embryo Twinning (AET)
- Somatic Cell Nuclear Transfer (SCNT)
Plant Cloning
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November 19, 2013
Protein Engineering
- Used to improve existing proteins,
such as enzymes, antibodies and cell
receptors, and to create proteins not
found in nature
- Used in drug development, food
processing and industrial
manufacturing
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November 19, 2013
1. Baking
> Flour consists of gluten, starch, non-starch
polysaccharides, lipids and trace amounts of
minerals.
>Optimize a combination of lower dosages of
enzymes to achieve optimum dough consistency,
stability, and bread quality.
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November 19, 2013
Fungal alpha-amylase:
Maximizes the fermentation process to obtain An even crumb structure
and a high loaf volume.
Glucose oxidase:
Oxidizes free sulphydryl groups in gluten to smaller crumb cells and a
silkier texture elastic
Lipase:
Dough conditioning by producing more uniform, make weak dough
stronger and more and whiter crumb color.
Lipoxygenase:
Bleaching and strengthening dough
Xylanase:
Dough conditioning, Easier dough handling and improved crumb structure
Protease:
Weakens the gluten to provide the plastic properties required in dough for
biscuits
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November 19, 2013
Feed industry
> Enzyme supplements in feed increase
animal growth rate and performance and
decrease the potential environmental pollution
from animal fecal excretion
- Phytase
- Hemi-cellulose degrading enzyme, e.g.,
glucanase and xylanase.
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November 19, 2013
Biosensors
- Helps human knowledge of biology to combine
advances in microelectronics
- A biosensor is composed of a biological
component, such as a cell, enzyme or antibody,
linked to a tiny transducer [a device powered by
one system that then supplies power (usually in
another form) to a second system]
- Biosensors are detecting devices that rely on the

specificity of cells and molecules to identify and
measure substances at extremely low
concentrations.
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November 19, 2013
Nanobiotechnology
Nanotechnology the study, manipulation and
manufacture of ultra-small structures and
machines made of as few as one molecule
Nanobiotechnology - involves developments in

nanotechnology and microbiology
Most appropriately, DNA, the information storage
molecule, may serve as the basis of the next
generation of computers.
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November 19, 2013
Microarrays
- is a hybridization of a nucleic acid sample (target) to a
very large set of oligonucleotide probes, which are
attached to a solid support, to determine sequence or to
detect variations in a gene sequence or expression or for
gene mapping (NCBI)
- Used in analysing a vast number of samples

simultaneously
- Thousands of DNA or proteins are arrayed on glass

slides to create DNA or protein chips
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November 19, 2013
Types:
1.DNA Microarrays
2.Protein Microarrays
3.Tissue Microarrays
4.Whole-cell microarrays
5.Small-molecule microarrays
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November 19, 2013
Movie Clip Available at
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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November 19, 2013
BIBLIOGRAPHY
Bertoni, G., HARDIN, J.F., and Kliensmith, L.J., 2012.

Beckers World of Cell. 8th ed. Pearson Education, Inc. Appendix,
A-1-29.
Karp, Gerald. 2010. Cell and Molecular Biology: Concepts and
Experiments. 6th ed. John Wiley & Sons, Inc. Chapter 18. pp.
715-730.
Paras Yadav, Annu Yadav, P. Kumar, J.S. Arora, T.K.Datta, S. De,
S.L. Goswami, Mukesh Yadav, Shalini Jain, Ravinder Nagpal
and Hariom Yadav. PPT Presentation: Basics of Cell Culture.
http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.
cfm?coSiteNavigation_allTopic=1
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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November 19, 2013
Thank you for listening.

^_^
Page 49
November 19, 2013

(Chapter III) RDT Techniques and Apps

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Previous Topics Discussed:

1. Central Dogma of Biology

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

Amplification & Screening

November 19, 2013

Amplification & Screening

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

Movie Clip Available at

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

YAC vector - about 10,000 bp

November 19, 2013

Bacterial artificial chromosome (BAC)

>One type of BAC facilitates the process of screening for

November 19, 2013

Screening DNA Libraries

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

Colony Hybridization Technique

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

DNA Fingerprinting by: Southern Blotting

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

November 19, 2013

Polymerase Chain Reaction

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

Sample computation for PCR preparation

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013

Suggested Cycling Parameters (iNtRON Biotechnology, Inc.)

PCR product size

RDT Techniques and Applications by Sarah May M. Querubin

Optional. Normally, 2-5min

November 19, 2013

RDT Techniques and Applications by Sarah May M. Querubin

November 19, 2013