Biotechnology and

Recombinant DNA

Chapter 9

Biotechnology and
Recombinant DNA
Biotechnology

The use of microbiological and biochemical

techniques to solve problems and produce
product
Recombinant DNA techniques

Methods used to manipulate DNA to intentionally

genetically alter organisms through genetic
engineering
Often to give organisms more useful traits

Fundamental Tools of Biotechnology

Basic components of molecular biologists

toolkit
Restriction enzymes
Gel electrophoresis
DNA probes
Primers

Fundamental Tools of Biotechnology

Restriction enzymes (Extracted from Bacteria)

Naturally occurring enzymes that cut DNA into

fragments

Cut in predictable and controllable manner

Generates pieces of DNA called restriction

fragments

These fragments can be joined to new fragments

Enzymes produce jagged cuts called sticky ends
Ends anneal together to form new strand

DNA ligase covalently joins fragments

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Fundamental Tools of Biotechnology

Gel electrophoresis

Used to separate DNA fragments according to

size

DNA is put into wells in gel

Gel subjected to current
DNA moves through the gel
Fragments are separated according to size
Large fragments remain high in the gel
Small fragments migrate lower

Gel must be stained to view DNA

Stained with ethidium bromide solution

Fundamental Tools of Biotechnology

DNA probes

Used to locate nucleotide sequences in DNA

or RNA
Probe is single-stranded piece of DNA tagged
with detectable marker

Location can be easily determined

Probe will hybridize to complementary

fragment of interest

Using a DNA probe to find the

colony with the gene of interest
Alignment Marks

Fundamental Tools of Biotechnology

Primers

Single stranded DNA fragments that bind

sequences of DNA
Used in in vitro DNA synthesis

Primer serves fragment for addition of DNA

nucleotides

Applications of
Genetic Engineering
Genetically engineered

bacteria

Genetic engineering
relies on DNA cloning
Process of producing
copies of DNA
Cloned DNA
generally combined
with carrier molecule
called cloning vector
Insures replication
of target DNA

Applications of
Genetic Engineering
Genetically engineered organisms have

variety of uses
Protein production
DNA production
Researching gene function and regulation
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Applications of
Genetic Engineering
Protein production

Produce commercially important proteins

Pharmaceutical proteins
Human insulin

Vaccines
Hepatitis B vaccine

Commercially valuable proteins

Chymosin An enzyme that catalyzes the coagulation
of milk used in the production of cheese

Applications of
Genetic Engineering
DNA production

Researches interested in acquiring available

sources of specific DNA fragments
Fragments used for

DNA study
Looking genomic characteristics

DNA vaccines
Looking at injecting DNA of pathogen to produce
immune response

Applications of
Genetic Engineering
Researching gene function and

regulation
Function and regulation can
be more easily study in
certain bacteria

E. coli used often due to

established protocols

Gene expression can be

studied by gene fusion

Joining gene being studied

to reporter gene
Reporter gene encodes
observable trait
Trait makes it possible
to determine changes
in gene

Applications of
Genetic Engineering
Genetically engineered eukaryotes
Yeast serve as important eukaryotic model for gene
function and regulation
Plant or animal that receive engineered gene termed
transgenic organism
Examples of genetically altered plants include
Pest resistant plants
Corn, cotton and potatoes

Herbicide resistant plants

Soybeans, cotton and corn

Plants with improved nutrient value

Rice

Plants as edible vaccines

Bananas and potatoes

Applications of
Probe Technologies
Variety of technology employ DNA probes

Colony blotting
Southern blotting (check for specific DNA in

electrophoresis samples)

Fluorescence in situ hybridization (FISH) (check

for specific DNA sequences in whole chromosomes)

DNA microarray

Applications of
Probe Technologies
Colony blotting

Used to detect specific DNA

sequences in colonies
grown in agar plates
Colonies are transferred in
place on nylon membrane
Colony blots are used to
determine which cells
contain genes of interest

Applications of
Probe Technologies
Southern blotting

Uses probes to detect

DNA sequences in
restriction fragments
separated using gel
electrophoresis
Application of
Southern blotting is
locating DNA
sequences similar to
ones being studied

Applications of
Probe Technologies
Fluorescence in situ hybridization (FISH)

Uses fluorescently labeled probes to detect

certain nucleotide sequences

Detects sequences inside intact cells

Specimens are viewed using fluorescence

microscopes
FISH can be used to identify specific
properties of bacteria

Mycobacterium tuberculosis in sputum sample

Applications of
Probe Technologies
DNA microarray

technologies

DNA arrays are solid

supports with fixed patterns
of different single stranded
DNA fragments attached
Enables researches to
screen sample for
numerous sequences
simultaneously

Applications for
DNA Sequencing
Knowing DNA sequence of particular cell

helps identify genetic alterations

Alterations that may result in disease

Sickle cell anemia

Due to single base-pair change in a gene

Cystic fibrosis
Caused by three base-pair deletion

Applications of
Polymerase Chain Reaction
Creates millions of

copies of given region of

DNA in matter of hours

Technique exploits
specificity of primers
Allows for selective
replication of chosen
regions
Termed target DNA

Large amounts of
DNA can be
produced from very
small sample

Care must be taken to prevent

contamination with external

source of target DNA
Basis for false-positive test
results
PCR Presentation

Techniques Used in Genetic Engineering

Obtaining DNA to be cloned
Generally through cell lysis
Generating a recombinant molecule

Restriction enzymes and ligases are used to

create a recombinant molecule

Introducing recombinant molecule into new host

Host acts as an incubator for DNA replication
DNA-mediated transformation often used to get
DNA into host

Techniques used in Probe Technologies

Probe technologies include

Colony blotting
Southern blotting
FISH
Microarray technology

Techniques used in Probe Technologies

Techniques in colony and Southern blotting

Blotting steps transfer sample to nylon

membrane
Probe is added

Probe hybridizes with complementary sequence

Process is used to locate positions of

hybridized probe

Techniques used in Probe Technologies

Techniques used in FISH

Sample preparation is critical

Methods used depend on type of organism

Specimen is applied to glass slide

Fluorescent label is applied and incubated

Incubation allows for hybridization

Specimen is view with fluorescence

microscope

Techniques Used in DNA Sequencing

Dideoxychain termination
Elements for termination reaction include

Single-stranded DNA template

Primer that anneal to template
DNA polymerase
Each of the nucleotide bases
One of these bases is labeled with marker for detection

Dideoxynucleotides
Like deoxynucleotide counterparts but lack 3 OH
Incorporation causes chain termination

Special gel electrophoresis used to separate DNA

fragments by size

Techniques Used in DNA Sequencing

Automated DNA sequencing

Most automated systems

use fluorescent dyes to
detect newly synthesized
DNA
Gel electrophoresis used
to separate fragments
into colored bands
Laser used to detect
color differences

Order of color reflects

nucleotide sequence

Techniques Used in Polymerase Chain Reaction

Starting with double stranded DNA molecule, process

involves number of amplification cycles

PCR requires three step amplification cycle

Step 1: double stranded DNA denatured by heat

Step 2: primers anneal to complementary sequence of
target DNA and DNA synthesis occurs with heat stable
DNA polymerase
Step 3: duplication of target DNA

DNA is amplified exponentially