© life_edu lecture 4 some techniques in biotechnology issues in biotechnology: the way we work with...

© life_edu

Lecture 4

Some Techniques in Biotechnology

Issues in Biotechnology:The Way We Work With Life

Dr. Albert P. Kausch

life edu.us

The Mechanics of DNA

Issues in Biotechnology:Biotechnology, Our Society and Our Future

OnCampus LiveOnCampus LiveBCH 190, MIC 190, AFS 190, NRS 190, PLS 190BCH 190, MIC 190, AFS 190, NRS 190, PLS 190

OnLine BCH 190OnLine BCH 190

A Sweeping General Survey on Life and BiotechnologyA Public Access College Course

The University of Rhode Island

Kimberly Nelson

Issues in Biotechnology:The Way We Work With Life

Dr. Albert P. Kausch

life edu.us

© life_edu

A Sweeping General Survey on Life and Biotechnology

The University of Rhode Island

Issues in Biotechnology:The Way We Work With Life

Dr. Albert P. Kausch

life edu.us

BCH 190BCH 190Section I.

The Mechanics of Life and General Biotechnology

© life_edu

3. Atoms, Cells and the Flow of Life

4. Some Techniques in Biotechnology

A Sweeping General Survey on Life and BiotechnologyA Public Access College Course

The University of Rhode Island

Issues in Biotechnology:The Way We Work With Life

Dr. Albert P. Kausch

life edu.us

The Mechanics of DNA

© life_edu

Lecture 4

Some Techniques in Biotechnology

Issues in Biotechnology:The Way We Work With Life

Dr. Albert P. Kausch

life edu.us

The Mechanics of DNA

Tools of the TradeThe eppendorf tubeand the pipetmanare the standard stock and trade in the dailywork of a molecularbiologist

Tools of the TradeThe eppendorf tubeand the pipetmanare the standard stock and trade in the dailywork of a molecularbiologist

“On the body of the traditional P-Series pipet it says, in relief, “Gilson.” Warren Gilson, who earned his MD in 1940 at the Univ. of Wisconsin, invented and patented the mechanical basis for the popular adjustable pipet (US Patent No. 3,827,305, 1974), Nearly 40 yrs. After the patent, the Pipetman continues to be manufactured in France in a factory started by Gilson’s colleague, Eugene Marteau D’Autry. Shortly before Gilson’s patent issued Gilson sold the marketing and sales rights to Ken Rainin President of Rainin Instrument, because, Gilson says, ‘He was a good salesman.’”

“Eppendorf tubesand PipetmanFor the Gold Rush”

Innovative technologies become biotech products

Separation Techniques: The need to separate the components of Life

Precipitation/DissolutionFiltersCentrifugationAffinityBlotsMagneticsElectrophoresisEtc.

The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 2,000,000 G (approx 19 600 km/s²). Ultracentrifuges find important uses in molecular biology, biochemistry and polymer science, including separation of cellular structures and molecules.

Electro refers to the energy of

electricity. Phoresis, from the

Greek verb phoros, means

“to carry across.” Thus, gel

electrophoresis refers to the

technique in which molecules

are forced across a span of gel,

motivated by an electrical

current.

Gel Electrophoresis:the separation of molecules,DNA, RNA and proteinsby charge and size

What is a Gel?What is a Gel?

Agarose is a long chain of sugar molecules,

a polymer, derived from algae

used in electrophoresis to separate molecules

Two types of gel:• Agarose (horizontal type)

• Polyacrylamide (vertical type)

How are Gels Loaded and Run?How are Gels Loaded and Run?

Applications of Gel Electrophoresis

• DNA Fingerprinting

• DNA Recombinant Technology

• Forensics

• The Human Genome Project

DNA carries a net negative charge; it is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.

The gel matrix acts as a sieve for DNA molecules. Large molecules have difficulty getting through the holes in the matrix. Small molecules move easily through the holes. Because of this, large fragments will lag behind small fragments as DNA migrates through the gel.

As the separation process continues, the separation between the larger and smaller fragments increases.

• Molecular weight markers are often electrophoresed with DNA.• Molecular weight markers are usually a mixture of DNAs with known molecular weights.• Molecular weight markers are used to estimate the sizes of DNA fragments in a DNA sample.

Issues in Biotechnology

Gel electrophoresis is an important tool in molecular biology and biotechnology. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Gel electrophoresis allows for:

(A) the separation of biological molecules, including DNA, RNA and

proteins by their charge and size (B) all of the answers are correct (C) the identification of DNA markers now commonly used in forensics to implicate or exonerate persons accused of various crimes (D) the rapid visualization of the products of PCR (E) the acceleration of DNA into cells for genetic engineering purposes

The Techniques of The Techniques of Molecular BiotechnologyMolecular Biotechnology

Technology has created new Fields

DNA detection GenomicsDNA synthesis Bioinformatics

DNA sequencing Pharmacogenomics

DNA cloning Transgenics

Expression cassette Computationalconstruction Biology

RNA detection Population Genetics

Protein detection Proteomics

The Techniques of The Techniques of Molecular BiotechnologyMolecular Biotechnology

Technology has created new Fields

DNA detection GenomicsDNA synthesis Bioinformatics

DNA sequencing Pharmacogenomics

DNA cloning Transgenics

Expression cassette Computationalconstruction Biology

RNA detection Population Genetics

Protein detection Proteomics

DNA does not replicate spontaneously, but is facilitated by a group of proteins Interestingly, each of these proteins is

coded for in DNA they also replicate

Proteins Are Used to Copy DNA

Enzymes were discovered that cut DNA at specific sequences

And subsequently, enzymes were discovered that paste DNA together

The ability to cut and paste DNA allowed gene cloning

Plasmids are circular pieces of DNA found in some bacteria

Many copies per cell

Antibiotic resistance gene

Plasmids can be cut and pasted back together

Foreign genes can be inserted

How is a gene cloned?Foreign DNA (gene)is inserted into a plasmid that has a gene for antibiotic resistance

The plasmid is introduced into a bacterial cell and grown on the antibiotic

Only bacteria with the plasmid grow…the inserted gene is copied many times

Promoter Coding Sequence Terminator

Gene Construction

Cell specificityDevelopmental specificityStart transcription

Protein coding sequenceStop transcriptionMessage stability

Gene constructs can be moved into plants and the gene is expresseddriven by the promoter sequence

It is now possible to clone any gene from any

organism and move it into any other organism

Gene transfer from one organism to another is not new

Image of two species of bacteria transferring viralphage particles

Bacteria transfer genesto other bacteria and plants

Now in nature thereis another organismcapable of transferring DNA: we call that organisma human being

Tools and Techniques Tools and Techniques used in Biotechnologyused in Biotechnology

     No Walls

The Clear bead at the center changes everythingThere are no edges to my being now

I have heard it said that there is a windowThat opens from one mind to another

But where there are no wallsThere is no need for a window, or fitting a latch.

Rumi 1279 AD     

For those who are interested in taking this For those who are interested in taking this course for college credit through the course for college credit through the

University of Rhode Island; University of Rhode Island; For more information please contact:For more information please contact:

  Issuesinbiotechnology190@gmail.comIssuesinbiotechnology190@gmail.com

CreditsCredits

Lectures by: Lectures by:

Edited by:Edited by:

Video Produced by:Video Produced by:

Thank You to The University of Rhode Island Thank You to The University of Rhode Island and all of the students of Issues in and all of the students of Issues in

Biotechnology over the yearsBiotechnology over the years

Dr. Albert KauschDr. Albert Kausch

Dr. Albert Kausch Dr. Albert Kausch and Kimberly Nelsonand Kimberly Nelson

Thaddeus WeaverThaddeus Weaver