Biotechnology and Recombinant DNA

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 file:///N:/Biology/Power%20Points/20_Lectures_PP

T/media/20_03RestrictionEnzymes_A.swf



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


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


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 file:///N:/Biology/Power%20Points/20_Lectures

_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


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



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 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


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


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


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 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

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Biotechnology and Recombinant DNA