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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
http://w
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/lesson/bacgen.files/10-20.gif
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