DNA

Technology

Biotechnology alteration of organisms or their components with

specific applications in mind

Breeding strategies

Artificial selection

Mutations

Genetic engineering - Direct manipulation of genes

- Recombinant DNA technology

https://sites.google.com/site/asoftsb/home/methods-of-selective-breeding/examples

http://cal.vet.upenn.edu/projects/genetic/inbreed/images/diagram2.gif

Basic DNA Techniques

Identification and isolation of GENE

OF INTEREST

Purification and fragmentation

using RESTRICTION

ENZYMES

Incorporation of DNA into CLONING

VECTORS (i.e. plasmids)

Incorporation of plasmids into bacterial host

through TRANSFORMATION

SELECTION of hosts carrying

desired recombinant gene

GROWING of selected host cells in culture medium

Gene cloning

• amplification of genes

• production of protein products

Restriction

Enzymes

• aka restriction

endonucleases

• produced by

Bacteria/Archaea

• cut at (restriction

sites) that are 4-8

base pairs long

• long DNA fragments

can be cut to

produce restriction

fragments

• e.g. EcoRI

(G*AATTC)

Constructing

recombinant

DNA with the

aid of

restriction

enzymes

A comprehensive list is available at

http://www.sciencegateway.org/RES

/index.html

DNA Insertion

• conditions that

allow host cells to

take up the vector

– high salt

concentration

– high temperature

(~42oC)

– electric pulse

– microinjection

Selection of transgenic host cells • recombinant molecules

must be separated from molecules consisting of just donor or just plasmid DNA

• plasmid used contains genes for resistance against certain antibiotics

• large number of cells grown from single cell carrying recombinant plasmid

• w/in hrs, the a whole culture of cells containing the recombinant DNA

http://filebox.vt.edu/users/chagedor/biol_4684/Methods/vector.gif

Genomic Library

• Complete

set of

plasmid-

containing

cell clones

Using cDNA

• difference

between

prokaryotes &

eukaryotic DNA

• DNA copied from

mature mRNA

transcript by

reverse

transcription

• cDNA library

Basic DNA Techniques

• DNA Amplification

– method of creating multiple copies

of a particular segment of DNA

– e.g. growing recombinant cells

polymerase chain reaction

(PCR)

Polymerase

Chain Reaction

Makes use of Taq

polymerase

Steps:

1. Denaturation

2. Annealing

3. Extension

Each PCR cycle of

heating and cooling

the DNA mixture

doubles the number

of DNA molecules.

Basic DNA Techniques

Gel Electrophoresis

technique used to

distinguish DNA

molecules

applied electric field

forces DNA to migrate

through a medium and

distance themselves

from one another by

length

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Basic DNA Techniques

• DNA Sequencing

– Sanger method -

for determining

the nucleotide

sequence of a

piece of DNA

– many copies are

needed (by cloning

or PCR)

QUIZ 2 (by pair)

1. Describe 1 natural way to manipulate DNA.

2. Differentiate between biotechnology and

recombinant DNA technology.

3. What are restrictzion enzymes?

4. What is the role of ligase in DNA

recombination?

5. How can we ensure that only the host cell with

the gene of interest will be cloned?

6. What enzyme is needed in creating cDNA?

7. What are the three steps in PCR?

8. How does gel electrophoresis separate DNA of

different lengths?

9. What is the use of fluorescently-labeled bases

in DNA sequencing?