[chapter iii] rdt techniques and apps (1)

Previous Topics Discussed:

1. Central Dogma of Biology

2. Recombinant DNA Technology

RDT Techniques and Applications by Sarah May M. Querubin November 19, 2013

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


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.


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)



Movie Clip Available at

http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cf

m?coSiteNavigation_allTopic=1

Isolation of gene

Genetic/Genomic Library

- a collection of

recombinant vector

clones; each clone

carrying a particular

segment coming from the

initial genome


Isolation of gene

cDNA Libraries

- A collection of

DNA molecules

reverse transcribed

from the mRNAs in

a particular cell-

type


cDNA Preparation


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


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.


Screening DNA Libraries

-to facilitate easier step in obtaining

DNA materials needed for genetic

engineering


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


DNA Fingerprinting by: Southern Blotting

RFLPs


PAGE

Polyacrylamide

Gel Electrophoresis


SDS-PAGE


Polymerase Chain Reaction

(PCR)

Applications of PCR:

Amplifying DNA for cloning for

analysis

Testing for specific DNA sequences

of viruses

Comparing DNA molecules


C1 V1 Vf X26 tubes

PCR Buffer 10x 2.5µL 1x 65

MgCl2 25mM 2.5µL 50 x 10 -6 65

dNTP 10mM 2.5µL 25 x 10-6 65

i-TAQ 5 units/µL 0.2µL 1 unit 5.2

ddH2O 15.3µL 397.8

Primer 1.0µL 26

Template

Total

1.0µL

25µL

26

650µL

Sample computation for PCR preparation


PCR Cycle Temp. PCR product size

100-500bp 500-1000bp 1kb-5kb

Initial denaturation 94ºC 2min 2min 2min

30-40

cycles

Denaturation 94ºC 20sec 20sec 20sec

Annealing 50-65ºC 10sec 10sec 20sec

Extension 65-72ºC 20-30sec 40-50sec 1min/kb

Final Extension 72ºC Optional. Normally, 2-5min

Suggested Cycling Parameters (iNtRON Biotechnology, Inc.)



http://www.nature.com/scitable/content/ne

0000/ne0000/ne0000/ne0000/5471131/PC

R_machine_2.jpg

OTHER TECHNIQUES AND APPLICATIONS

OF DNA TECHNOLOGY


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

MAbs + toxin = therapeutic compounds RDT Techniques and Applications by Sarah May M. Querubin November 19, 2013


Hybridomas

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.


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


>Choice of media depends on the type of cell being cultured

>Asceptic Techniques

http://www.foodprocessing-

technology.com/contractor_im

ages/biomerieux-australia/3-

culture-media.jpg

http://www.rnd

systems.com/r

esources/imag

es/6227.jpg


Applications of Cell Culture

1. Model systems for Studying basic cell biology, interactions between disease- causing 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

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


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


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.


• 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


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.


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.


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.”


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


Types:

1.DNA Microarrays 2.Protein Microarrays 3.Tissue Microarrays 4.Whole-cell microarrays 5.Small-molecule microarrays



Movie Clip Available at

http://www.bio.davidson.edu/courses/genomics/chip/chip.html

BIBLIOGRAPHY

Bertoni, G., HARDIN, J.F., and Kliensmith, L.J., 2012.

Becker’s 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


Thank you for listening.

^_^


[chapter iii] rdt techniques and apps (1)

Documents

screening rdt techniques

segregated rdt techniques

recombinant dna vector

rdt steps

xgal rdt techniques

initial genome rdt techniques

recombinant bacteria

bp of foreign dna