Biotechnology and Recombinant DNA

gooseliverBiotechnology

Oct 22, 2013 (3 years and 10 months ago)

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Biotechnology and
Recombinant DNA

Chapter 9


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

Biotechnology and

Recombinant DNA

Fundamental Tools of Biotechnology


Basic components of molecular biologist’s
“toolkit”


Restriction enzymes


Gel electrophoresis


DNA probes


Primers


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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T/media/20_03RestrictionEnzymes_A.swf


Fundamental Tools of Biotechnology

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


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


Primers


Single stranded DNA fragments that bind
sequences of DNA


Used in
in vitro

DNA synthesis


Primer serves fragment for addition of DNA
nucleotides

Fundamental Tools of Biotechnology

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


Genetically engineered organisms have
variety of uses


Protein production


DNA production


Researching gene function and regulation


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_PPT/media/20_04CloningAGene_A.swf

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

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


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

Genetic Engineering

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 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 Probe Technologies

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