chapter 8 recombinant dna technology 10/1/111mdufilho

Chapter 8

Recombinant DNA

Technology

10/1/11 1MDufilho

• Recombinant DNA Technology– Intentional modification of organisms’ genomes for

practical purposes

– Three goals – Eliminate undesirable phenotypic traits– Combine beneficial traits of two or more organisms– Create organisms that synthesize products humans

need

10/1/11 2MDufilho

Figure 8.1 Overview of recombinant DNA technologyBacterial cell

Bacterialchromosome

Plasmid

Gene of interest

DNA containinggene of interest

Isolate plasmid.

Enzymatically cleaveDNA into fragments.

Isolate fragmentwith the gene ofinterest.

Insert gene into plasmid.

Insert plasmid and gene intobacterium.

Culture bacteria.

Harvest copies ofgene to insert intoplants or animals

Harvest proteinscoded by gene

Eliminateundesirablephenotypictraits

Produce vaccines,antibiotics,hormones, orenzymes

Createbeneficialcombinationof traits

10/1/11 3MDufilho

The Tools of Recombinant DNA Technology

• Mutagens– Physical and chemical agents that produce

mutations– Scientists utilize mutagens to

– Create changes in microbes’ genomes to change phenotypes

– Select for and culture cells with beneficial characteristics

– Mutated genes alone can be isolated

10/1/11 4MDufilho


• The Use of Reverse Transcriptase to Synthesize cDNA

– Isolated from retroviruses

– Uses RNA template to transcribe molecule of cDNA

– Easier to isolate mRNA molecule for desired protein first

– mRNA of eukaryotes has introns removed – Allows cloning in prokaryotic cells

10/1/11 5MDufilho


• Synthetic Nucleic Acids– Molecules of DNA and RNA produced in cell-

free solutions

– Uses of synthetic nucleic acids– Elucidating the genetic code– Creating genes for specific proteins– Synthesizing DNA and RNA probes to locate

specific sequences of nucleotides– Synthesizing antisense nucleic acid molecules

10/1/11 6MDufilho


• Restriction Enzymes– Bacterial enzymes that cut DNA molecules only at

restriction sites

– Categorized into two groups based on type of cut– Cuts with sticky ends– Cuts with blunt ends

10/1/11 7MDufilho

Figure 8.2 Actions of restriction enzymes-overview

10/1/11 8MDufilho


ANIMATION Recombinant DNA Technology

10/1/11 9MDufilho


• Vectors– Nucleic acid molecules that deliver a gene into

a cell

– Useful properties– Small enough to manipulate in a lab– Survive inside cells– Contain recognizable genetic marker– Ensure genetic expression of gene

– Include viral genomes, transposons, and plasmids

10/1/11 10MDufilho

Figure 8.3 Producing a recombinant vectorAntibioticresistancegene

Restrictionsite

mRNA for humangrowth hormone (HGH)

Reversetranscription

Plasmid (vector)

cDNA for HGH

Restrictionenzyme

Restrictionenzyme

Sticky ends

Gene for humangrowth hormone

Ligase

Recombinant plasmid

Introduce recombinantplasmid into bacteria.

Recombinantplasmid

Bacterialchromosome

Inoculate bacteriaon media containingantibiotic.

Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.10/1/11 11MDufilho


• Gene Libraries– A collection of bacterial or phage clones

– Each clone in library often contains one gene of an organism’s genome

– Library may contain all genes of a single chromosome

– Library may contain set of cDNA complementary to mRNA

10/1/11 12MDufilho

Figure 8.4 Production of a gene library-overview

Genome

Isolate genomeor organism.

Generate fragments usingrestriction enzymes.

Insert each fragmentinto a vector.

Introduce vectorsinto cells.

Culture recombinant cells;descendants are clones.

10/1/11 13MDufilho

• Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)

– Large number of identical molecules of DNA produced in vitro

– Critical to amplify DNA in variety of situations– Epidemiologists use to amplify genome of

unknown pathogen – Amplified DNA from Bacillus anthracis spores in

2001 to identify source of spores

10/1/11 14MDufilho

Techniques of Recombinant DNA Technology

• Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)

– Repetitive process consisting of three steps– Denaturation– Priming– Extension

– Can be automated using a thermocycler

10/1/11 15MDufilho


ANIMATION Polymerase Chain Reaction: Components

10/1/11 16MDufilho


.

ANIMATION PCR: The Process

10/1/11 17MDufilho


• Selecting a Clone of Recombinant Cells– Must find clone containing DNA of interest

– Probes are used

10/1/11 18MDufilho


• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot

– Gel electrophoresis– Separates molecules based on electrical charge, size,

and shape– Allows scientists to isolate DNA of interest– Negatively charged DNA drawn toward positive

electrode– Agarose makes up gel; acts as molecular sieve– Smaller fragments migrate faster than larger ones– Determine size by comparing distance migrated to

standards10/1/11 19MDufilho

Figure 8.6 Gel electrophoresis-overview

10/1/11 20MDufilho


• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot

– Southern blot– DNA transferred from gel to nitrocellulose membrane– Probes used to localize DNA sequence of interest– Northern blot: used to detect RNA

– Uses of Southern blots– Genetic “fingerprinting” – Diagnosis of infectious disease– Demonstrate incidence and prevalence of organisms

that cannot be cultured10/1/11 21MDufilho

Figure 8.7 The Southern blot technique-overview

DNA molecules

Restriction enzymes

Restriction fragments

Use gel electrophoresis to separatefragments by size; denature DNAinto single strands with NaOH.

Side view

DNA

DNA bandsGel

Nitrocellulosemembrane

Absorbentmaterial

The DNA fragmentsare invisible to theinvestigators atthis stage.

Electrophoresisgel

Nitrocellulosemembrane

Absorbentmaterial

Nitrocellulose membranewith DNA fragments atsame locations as in gel(still invisible) is baked topermanently affix DNA.

Add radioactive probescomplementary to DNAnucleotide sequenceof interest.

Probes bind to DNAof interest.

Incubate with film; radiation exposes film.Develop film.

Developed film

10/1/11 22MDufilho


• DNA Microarrays– Consist of molecules of immobilized single-

stranded DNA

– Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences

– Variety of scientific uses of DNA microarrays– Monitoring of gene expression– Diagnosis of infection– Identification of organisms in an environmental

sample10/1/11 23MDufilho

Figure 8.8 DNA microarray-overview

10/1/11 24MDufilho


• Inserting DNA into Cells– Goal of DNA technology is insertion of DNA into cell

– Natural methods – Transformation– Transduction– Conjugation

– Artificial methods– Electroporation– Protoplast fusion– Injection: gene gun and microinjection

10/1/11 25MDufilho

Figure 8.9a Artificial methods of inserting DNA into cells: electroporation

Chromosome

Electroporation

Pores in wall and membrane

Competent cell

Electricalfield applied

DNA fromanother source

Cell synthesizesnew wall

Recombinant cell

10/1/11 26MDufilho

Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion

Cell walls

Protoplast fusion

Polyethyleneglycol

Protoplasts

Enzymes removecell walls

Fused protoplasts

Recombinant cellNew wall

Cell synthesizesnew wall

10/1/11 27MDufilho

Figure 8.9c Artificial methods of inserting DNA into cells: gene gun

Gene gun

Protoplasts

Nylonprojectile

Nylonprojectile

Blank .22caliber shell

DNA-coated beads

Vent

Target cell

Plate to stopnylon projectile

10/1/11 28MDufilho

Figure 8.9d Artificial methods of inserting DNA into cells: microinjection

Microinjection

Target cell

Suction tubeto hold targetcell in place

Target cell’snucleus

Micropipettecontaining DNA

10/1/11 29MDufilho

Applications of Recombinant DNA Technology

• Genetic Mapping– Locating genes on a nucleic acid molecule

– Provides useful facts concerning metabolism, growth characteristics, and relatedness to others

• Locating Genes– Until 1970, genes identified by labor-intensive

methods

– Simpler and universal methods now available

– Restriction fragmentation

– Fluorescent in situ hybridization (FISH)10/1/11 30MDufilho

Figure 8.10 FISH

10/1/11 31MDufilho


• Environmental Studies– Most microorganisms have never been grown in a

laboratory

– Scientists know them only by their DNA fingerprints

– Allowed identification of over 500 species of bacteria from human mouths

– Determined that methane-producing archaea are a problem in rice agriculture

10/1/11 32MDufilho


• Pharmaceutical and Therapeutic Applications– Protein synthesis

– Creation of synthetic peptides for cloning

– Vaccines– Production of safer vaccines– Subunit vaccines– Genes of pathogens introduced into common fruits

and vegetables– Injecting humans with plasmid carrying gene from

pathogen– Humans synthesize pathogen’s proteins

10/1/11 33MDufilho


• Pharmaceutical and Therapeutic Applications– Genetic screening

– DNA microarrays used to screen individuals for inherited disease caused by mutations

– Can also identify pathogen’s DNA in blood or tissues

– DNA fingerprinting– Identifying individuals or organisms by their unique

DNA sequence

10/1/11 34MDufilho

Figure 8.12 DNA fingerprinting

10/1/11 35MDufilho


• Pharmaceutical and Therapeutic Applications– Gene therapy

– Missing or defective genes replaced with normal copies

– Some patients’ immune systems react negatively

– Medical diagnosis– Patient specimens can be examined for presence of

gene sequences unique to certain pathogens

– Xenotransplants– Animal cells, tissues, or organs introduced into

human body10/1/11 36MDufilho

The Ethics and Safety of Recombinant DNA Technology

– Supremacist view: humans are of greater value than animals

– Long-term effects of transgenic manipulations are unknown

– Unforeseen problems arise from every new technology and procedure

– Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms

– Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

10/1/11 37MDufilho


• Studies have not shown any risks to human health or environment

• Standards imposed on labs involved in recombinant DNA technology

• Can create biological weapons using same technology

© 2012 Pearson Education Inc.

10/1/11 38MDufilho


• Ethical Issues– Routine screenings?

– Who should pay?

– Genetic privacy rights?

– Profits from genetically altered organisms?

– Required genetic screening?

– Forced correction of “genetic abnormalities”?

© 2012 Pearson Education Inc.

10/1/11 39MDufilho

chapter 8 recombinant dna technology 10/1/111mdufilho

Documents