Ch 20: DNA TechnologyCh 20: DNA Technology

• The mapping and sequencing of the human genome has been made possible by advances in DNA technology.

• Human genome project

• We are now developing techniques for making recombinant DNA• Genes from two different sources - often different

species - are combined into the same molecule.

• DNA technology has launched a revolution in biotechnology.• The manipulation of organisms or their

components to make useful products.

• DNA technology is now applied in areas ranging from agriculture to criminal law, but its most important achievements are in basic research.

Introduction

• Recombinant DNA– Taking DNA from two sources and

combining then into one molecule.– Occurs naturally in viral transduction,

bacterial transformation, and conjugation

• Biotechnology (genetic engineering)– Engineering genes in the Lab

Recombinant DNA

Many tools and techniques have been developed to manipulate and engineer

genes.

1. Restriction Enzymes2. Gel Electrophoresis

-Restriction Fragment Polymorphisms

3. DNA Probe4. Polymerase Chain Reaction5. Complementary DNA

Restriction Enzymes• Discovered in the 1960s• Extracted from bacteria

– Used to fend off bacteriophages– Appear to serve a host-defense role– Protect own DNA by methaylation of adenines &

cytosines• REs cut DNA at specific sites called recognition

sequences or sites.– Leaving fragments of DNA

• Scientists have isolated 100s of REs– Named for the bacteria in which they were found.

• EcoRI• BamHI• HindIII

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NamingRestriction enzymes are named based on the bacteria in which they are isolated in the following manner:

E Escherichia (genus)

co coli (species)

R RY13 (strain)

I First identified Order ID'd in bacterium

Enzyme Organism from which derived Target sequence

(cut at *)5' -->3'

Ava I Anabaena variabilis C* C/T C G A/G G

Bam HI Bacillus amyloliquefaciens G* G A T C C

Bgl II Bacillus globigii A* G A T C T

Eco RI Escherichia coli RY 13 G* A A T T C

Eco RII Escherichia coli R245 * C C A/T G G

Hae III Haemophilus aegyptius G G * C C

Hha I Haemophilus haemolyticus G C G * C

Hind III Haemophilus inflenzae Rd A* A G C T T

Hpa I Haemophilus parainflenzae G T T * A A C

Kpn I Klebsiella pneumoniae G G T A C * C

Mbo I Moraxella bovis *G A T C

Mbo I Moraxella bovis *G A T C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

SstI Streptomyces stanford G A G C T * C

• The restriction sites are often a symmetrical series of four to eight bases on both strands running in opposite directions.

• If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’.

• Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.

• Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme.

CLIP

• Restriction enzymes cut covalent phosphodiester bonds of both strands• often in a staggered

way creating single-stranded ends, sticky ends.

• These extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme. •These DNA fusions can be made permanent by DNA

ligase which seals the strand by catalyzing the formation of phosphodiester bonds.

Must use same restriction enzyme on both.

Setting up a simple restriction digestion:

1. DNA: Reliable cleavage by restriction enzymes requires DNA that is free from contaminants such as phenol or ethanol. Excessive salt will also interfere with digestion by many enzymes, although some are more tolerant of that problem.

2. An appropriate buffer: Different enzymes cut optimally in different buffer systems, due to differing preferences for ionic preferences for ionic strengthstrength and major cation. When you purchase an enzyme, the company almost invariably sends along the matching buffer as a 10X concentrate.

3. The restriction enzyme!

Restriction Enzyme animation

Animation #2Cloning a Gene

Bacteria DNA that has DNA from another

organism spliced in to it.

Gel Electrophoresis• Method of rapidly analyzing and comparing

genomes.

Restriction Fragment Length Polymorphisms (RFLPs)

• The restriction pattern is different for every organism.

• This is why you get different banding patterns.

Gel Electrophoresis

DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA

AgarAgar is an unbranched polysaccharide obtained from the cell walls of some species of red algae or seaweed

Rate of movement depends on size, electrical charge, and other physical properties of the macromolecules.

• Separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel.

Simulation

• Because DNA has a negative charge, it moves toward the opposite side.

• Smaller fragments move greater distances

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Animation

We start by adding the restriction enzyme to each of the three samples to produce restriction fragments.

• We then separate the fragments by gel electrophoresis.

• Southern blotting (Southern hybridization) allows us to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size.

• Southern blotting -method in molecular biology of enhancing the enhancing the result of an agarose gel electrophoresis by marking specific DNA result of an agarose gel electrophoresis by marking specific DNA sequencessequences

• This also denatures the DNA fragments.

• Bathing this sheet in a solution containing our probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest and we can visualize bands containing the label with autoradiography.

We can tie together several molecular techniques to compare DNA samples from three individuals

Animation

• Common method of creating copies of specific fragments of DNA

• PCR rapidly amplifies a single DNA molecule into many billions of molecules

Polymerase Chain Reaction

(PCR); Making copies

Animation

Complementary DNA (cDNA)

• When scientists clone a human gene in a bacterium, the introns present introns present a problema problem.

• Bacteria lack introns and have no way to cut them out.

Animation

• In order to clone a human gene in a bacterium, the introns need to be removed.

• The genes is allowed to be transcribed and fully processed into mRNA.

• Reverse transcriptase is added to the mRNA and DNA copies are made.

• The DNA made by this process is called cDNA.

Complementary DNA (cDNA)

DNA probe; DNA Tagging

• A DNA probe is a radioactively labeled single strand of nucleic acid molecule used to tag a specific sequence in a DNA sample.

• The probe bonds to a complementary sequence wherever it occurs.

• Can identify genetic defects

Animation

Gene Therapy

Cloning

Genetically Modified Organisms

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Transgeneic Organisms CLIP

Transgenic Organisms 7272

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Transgenic Tobacco, from 1986. This is an ordinary photographic image of a tobacco plant engineered to express a firefly gene which produces luciferase.

Plasmids as vectors

• Lab on Fri

• Animation

Golden Rice23 times more Vitamin A

Called a Transgenic Organism

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•Researchers use recombinant DNA technology to analyze genetic changes. •They cut, splice together, and insert the modified DNA molecules from different species into bacteria or another type of cell that rapidly replicates and divides. •The cells copy the foreign DNA right along with their own DNA. •An example of this is the gene for human insulin inserted into a bacterium. This is how human insulin is mass produced.

•Not only does genetic engineering have applications in medicine and the environment, it also has uses in industry and agriculture. •Sheep are used in the production of alpha-1 antitrypsin, which is used in the treatment of emphysema. •Goats are also producing the CFTR protein used in the treatment of cystic fibrosis.

In the plant world, the buds of cotton plants are vulnerable to worm attacks. The buds of a modified cotton plant resist these worms, resulting in increased cotton production.These gene insertions are ecologically safer than pesticides. They affect only the targeted pest.

Plant biologists have used DNA technology to produce plants with many desirable traits. These include increased disease resistance, herbicide resistance, and increased nutritional content.

Scientists today have developed Scientists today have developed genetically altered bacteria. genetically altered bacteria. Among them are strains of bacteria Among them are strains of bacteria that that

eat up oil spillseat up oil spillsmanufacture alcohol and other manufacture alcohol and other chemicalschemicalsprocess minerals. process minerals.

There is concern about possible risks There is concern about possible risks to the environment and the general to the environment and the general population as genetically engineered population as genetically engineered bacteria are introduced.bacteria are introduced.