6.1 - 6.26.1 - 6.2 Biotechnological Tools and Biotechnological Tools and

Techniques of Genetic Techniques of Genetic EngineeringEngineering

Genetic EngineeringGenetic EngineeringWhat is it used for?What is it used for?

altering the sequence of DNA molecules to:

- investigate genetic disorders- produce drugs (eg. insulin somatotropin)

- - produce of GMOs

Genetic Engineering PioneersGenetic Engineering Pioneers

1970s – Stanley Cohen (plasmids) and Herbert Boyer (restriction enzymes)– Developed experiments for selecting,

recombining and transforming new genes into bacteria

– Techniques are still used in molecular biology labs to transfer new genes

– one of the most important discoveries in biomedical research

– 1978 – “Genentech” biotechnology company produced somatostatin

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SomatropinSomatropin A drug that is identical to human

growth hormone (somatotropin)– treats growth deficiency from

dwarfism and Turner’s syndrome– was the first human hormone

produced by genetic engineering using bacteria to deliver new genes to deficient individuals

Recombinant DNARecombinant DNA

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory

methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources,

creating sequences that would not otherwise be found in biological

organisms.

Things to consider with this technology:– where to cut DNA and where to

insert new gene material– how to put DNA back together

SomatropinSomatropinproductionproduction

Excise human growth hormone gene using restriction endonucleases

Signal sequence is removed

Insert gene after the lac promoter in a plasmid transformed into E. coli cells

E. coli are grown in media containing IPTG (isopropyl thiogalactoside)

Somatropin production (contSomatropin production (cont’’d)d) IPTG is an inducer of the lac promoter but is not

consumed by E.coli Control production of hormone

– Presence of IPTG somatropin production– Absence of IPTGno somatropin production

Somatropin is then isolated from bacteria and sold to patients

lacZ lacY lacAsomatropin

RNA polymerase

lacI protein

IPTG

mRNA

Somatropin, ß-galactosidase, permease, transacetylase are synthesized

Tools of BiotechnologyTools of Biotechnology1. 1. Restriction EndonucleasesRestriction Endonucleases

Also known as restriction enzymes Essentially are molecular scissors Recognize a specific DNA sequence and cut the

strands at a particular position or “recognition site” Isolated and purified only from bacteria

– named after bacteria the enzyme originates from– ie. EcoRI Escherichia coli, strain R, 1st r.e.

isolated

HindII Haemophilus influenzae, strain Rd, 2nd r.e.

Restriction Endonucleases: Recognition Restriction Endonucleases: Recognition sitesite

Each restriction endonuclease recognizes aspecific ‘recognition’ site (DNA sequence)

Usually 4-8 base pairs long, characterized by a complementary palindromic sequence

Bacteria Restriction Enzyme

Recognition Site

Escherichia coli EcoRI 5’-GAATTC-3’

3’-CTTAAG-5’

Haemophilus parainfluenzae

HindIII 5’-AAGCTT-3’3’-TTCGAA-5’

Arthrobacter luteus

AluI 5’-AGCT-3’3’-TCGA-5’

Restriction Endonucleases: Restriction Endonucleases: FunctionFunction

scans DNA and binds to its specific recognition sequence

disrupts the phosphodiester bonds between particular nucleotides through a hydrolysis reaction

Hydrogen bonds of the complementary base pairs in between the cuts are disrupted

Result: 2 DNA fragments

http://www.scq.ubc.ca/?p=249

Restriction Endonucleases: DNA Restriction Endonucleases: DNA Fragment EndsFragment Ends

Different DNA fragment ends are produced after digestion by different restriction enzymes– Sticky ends: DNA fragment ends with short single-

stranded overhangs (ie. EcoRI, HindIII)– Blunt ends: DNA fragment ends are fully base paired

(ie. AluI)

Bacteria Restriction enzyme

Recognition site After digestion by restriction enzyme

Escherichia coli EcoRI 5’-GAATTC-3’

3’-CTTAAG-5’

5’-G AATTC-3’

3’-CTTAA G-5’

Haemophilus parainfluenzae

HindIII 5’-AAGCTT-3’3’-TTCGAA-5’

5’-A AGCTT-3’3’-TTCGA A-5’

Arthrobacter luteus

AluI 5’-AGCT-3’3’-TCGA-5’

5’-AG CT-3’3’-TC GA-5’

Restriction Endonucleases: DNA Fragment Restriction Endonucleases: DNA Fragment Ends Ends

Palindrome

Restriction site

Fragment 1 Fragment 2

http://www.bio-rad.com/LifeScience/docs/Official_Crime_Scene_PowerPoint_Spring_2005_rev_B.ppt

Restriction Endonucleases: Length of Restriction Endonucleases: Length of recognition sitesrecognition sites

longer recognition sites result in fewer cuts.

– EcoRI 5’-GAATTC-3’ = ¼ × ¼ × ¼ × ¼ × ¼ × ¼ = 1/4096– AluI 5’-AGCT-3’ = ¼ × ¼ × ¼ ×¼ = 1/256

higher frequency of cuts – may cut gene into several fragments

lower frequency of cuts – may produce larger fragments than desired

Restriction Endonucleases: Restriction Endonucleases: MethylasesMethylases

Enzymes that add a methyl group to a nucleotide within a recognition site to prevent restriction endonucleases from cutting DNA

Helps distinguish between foreign (viral) DNA and the bacteria’s own DNA

Restriction Endonucleases:Restriction Endonucleases: DNA LigaseDNA Ligase

Enzyme that rejoins cut strands of DNA together by reforming a phosphodiester bond

DNA ligase joins sticky endsT4 DNA ligase (from T4 bacteriophage)

joins blunt ends

Sorting out DNA Sorting out DNA

2. 2. Gel ElectrophoresisGel ElectrophoresisTechnique used to separate charged

molecules based on their sizeDifferentiates one piece from other, Acts like a molecular sieve to sort out DNA

http://www.biotech.iastate.edu/ppt_presentations/html/Fingerprinting/StudentInstruction-gel/images/image08.jpg

http://www.solve.csiro.au/1105/img/sieve-bloke.jpg

Gel Electrophoresis: Gel Electrophoresis: DNA PreparationDNA Preparation

Restriction enzymes digest DNA into smaller fragments of different lengths

Different DNA samples are loaded into wells of the gel (agarose or polyacrylamide)

http://www.oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg

Gel Electrophoresis: Attraction Gel Electrophoresis: Attraction MigrationMigration

Negatively charged electrode is at the end where wells are located

Positively charged electrode at opposite endNegatively charged DNA migrate towards

positive end due to attraction

Gel Electrophoresis: Gel Electrophoresis: Rate of MigrationRate of Migration

Shorter/lighter DNA fragments migrate through gel faster since they can move through the pores in the gel more easily

Longer/heavier DNA fragments migrate through gel slower Rate of migration = 1/log(size) Different DNA fragment lengths are therefore separated

http://www.answers.com/topic/agarosegel-jpg

A = kilobase DNA ladder

B = uncut plasmid DNA

C = single digestion of the plasmid with EcoRI

D = single digestion with XhoI

E = double digestion - both EcoRI and XhoI.

A B C D E

Visualizing DNA FragmentsVisualizing DNA Fragments

Ethidium bromide is a fluorescent dye that makes DNA fragments visible

DNA fragments can then be isolated and purified

http://www.answers.com/topic/agarosegel-jpg

Gel Electrophoresis: Proteins too!Gel Electrophoresis: Proteins too!

Gel electrophoresis can also be used to separate proteins, usually using polyacrylamide gels

http://www.biotechlearn.org.nz/var/biotech/storage/images/multimedia/images/protein_electrophoresis/48251-4-eng-GB/protein_electrophoresis_medium.jpg

http://www.bio-link.org/vlab/Graphics/Tools/ProteinGel2.jpg

3. 3. PlasmidsPlasmidssmall, circular double-stranded DNA that

can enter and exit bacterial cellslack a protein coatindependent of bacterial chromosome1000-200,000 base pairs

3. Plasmids: 3. Plasmids: EndosymbiosisEndosymbiosis

Use host bacterial enzymes and ribosomes to replicate and express plasmid DNA

Carry genes that express proteins to protect bacteria against antibiotics & heavy metals

3. What are plasmids used for?3. What are plasmids used for? Foreign genes (ie. insulin) can be inserted into plasmids,

so bacteria can express gene and make its respective protein.

A higher ‘copy number’ of plasmids (number of individual plasmids) in bacteria results in larger number of gene copies and thus more of its respective protein is synthesized

PlasmidsPlasmids Restriction

endonucleases splice foreign genes into plasmids

DNA ligase reforms phosphodiester bond between the fragments, resulting in recombinant DNA

http://www.accessexcellence.org/RC/VL/GG/inserting.html

TransformationTransformationRefers to introduction of foreign DNA

(usually a plasmid) into a bacterium

Plasmids are used as a vector (vehicle) to introduce new genes into a host cell.

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Siegenthaler/fig2.gif

Transformation: Transformation: CompetenceCompetence Competent cell - bacterium that readily takes up foreign

DNA (ie. able to undergo transformation) Most cells are not naturally competent, but can be

chemically induced to become competent

–Calcium ion in calcium chloride stabilizes negatively charged phosphates on bacterial membrane

Transformation: Transformation: CompetenceCompetence