Biotechnology and Recombinant DNA


Oct 22, 2013 (4 years and 8 months ago)


Biotechnology and
Recombinant DNA

Chapter 9


The use of microbiological and biochemical
techniques to solve problems and produce

Recombinant DNA techniques

Methods used to manipulate DNA to intentionally
genetically alter organisms through genetic

Often to give organisms more useful traits

Biotechnology and

Recombinant DNA

Fundamental Tools of Biotechnology

Basic components of molecular biologist’s

Restriction enzymes

Gel electrophoresis

DNA probes


Restriction enzymes (
Extracted from Bacteria

Naturally occurring enzymes that cut DNA into

Cut in predictable and controllable manner

Generates pieces of DNA called restriction

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


Fundamental Tools of Biotechnology

Fundamental Tools of Biotechnology

Gel electrophoresis

Used to separate DNA fragments according to

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

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

Fundamental Tools of Biotechnology

Using a DNA probe to find the
colony with the gene of interest

Alignment Marks


Single stranded DNA fragments that bind
sequences of DNA

Used in
in vitro

DNA synthesis

Primer serves fragment for addition of DNA

Fundamental Tools of Biotechnology

Applications of

Genetic Engineering

Genetically engineered

Genetic engineering
relies on DNA cloning

Process of
producing copies of

Cloned DNA
generally combined
with carrier molecule
called cloning vector

Insures replication
of target DNA

Genetically engineered organisms have
variety of uses

Protein production

DNA production

Researching gene function and regulation


Applications of

Genetic Engineering

Protein production

Produce commercially important proteins

Pharmaceutical proteins

Human insulin


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

Applications of

Genetic Engineering

Researching gene function and

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

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


Plants as edible vaccines

Bananas and potatoes

Applications of

Genetic Engineering

Applications of

Probe Technologies

Variety of technology employ DNA probes

Colony blotting

Southern blotting (
check for specific DNA in
electrophoresis samples)

in situ

hybridization (FISH) (
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

Application of
Southern blotting is
locating DNA
sequences similar to
ones being studied

Applications of

Probe Technologies

in situ

hybridization (FISH)

Uses fluorescently labeled probes to detect
certain nucleotide sequences

Detects sequences inside intact cells

Specimens are viewed using fluorescence

FISH can be used to identify specific
properties of bacteria

Mycobacterium tuberculosis

in sputum sample

Applications of

Probe Technologies

DNA microarray

DNA arrays are solid
supports with fixed patterns
of different single stranded
DNA fragments attached

Enables researches to
screen sample for
numerous sequences

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

Technique exploits
specificity of primers

Allows for selective
replication of chosen

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

Basis for false
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

mediated transformation often used to get
DNA into host

Techniques used in Probe Technologies

Probe technologies include

Colony blotting

Southern blotting


Microarray technology

Techniques in colony and Southern blotting

Blotting steps transfer sample to nylon

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

Techniques used in Probe Technologies

Techniques Used in DNA Sequencing

Dideoxychain termination

Elements for termination reaction include

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


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

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