6.1 Biotechnological Tools and 6.1 Biotechnological Tools and TechniquesTechniques

Recombinant DNA & Gel electrophoresis

Recombinant DNARecombinant DNA

Cutting DNA fragments from different sources and recombining them together

Purpose–To investigate genetic disorders–Production of drugs (ie. insulin)

Cutting DNA fragments from different sources and recombining them together

What complications do you What complications do you foresee?foresee?

Consider:– The size of DNA– Where to cut?– How to put back together?

1. Restriction Endonucleases1. Restriction Endonucleases

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

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

– Name reflects which bacteria the enzyme originates– ie. EcoRI Escherichia coli, strain R, 1st r.e. isolated

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

1. Restriction Endonucleases: 1. Restriction Endonucleases: Recognition siteRecognition site

Each restriction endonuclease recognizes its own specific recognition site (specific 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’

1. Restriction Endonucleases: 1. 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

1. Restriction Endonucleases: 1. Restriction Endonucleases: DNA Fragment EndsDNA Fragment 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’

1. Restriction Endonucleases: 1. Restriction Endonucleases: DNA Fragment Ends DNA Fragment Ends (continued)(continued)

Palindrome

Restriction site

Fragment 1 Fragment 2

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

Animation

How do we control the snips?How do we control the snips?

Consider:– What about the organisms own DNA?– Frequency of recognition sequences within the

DNA sequence

1. Restriction Endonucleases: 1. Restriction Endonucleases: Length of recognition sitesLength of recognition sites

Longer recognition sites result in lower frequency of 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 large fragments than desired

1. Restriction Endonucleases: 1. Restriction Endonucleases: MethylasesMethylases

Enzymes that add a methyl group to a nucleotide in a recognition site to prevent restriction endonuclease from cutting DNA

Distinguishing between foreign (viral) DNA and bacteria’s own DNA

1. Restriction Endonucleases: 1. 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

How do we sort out the DNAHow do we sort out the DNA

DNA is chopped into many pieces

How to differentiate one piece from other

2. Gel Electrophoresis2. Gel Electrophoresis

Technique used to separate charged molecules based on their size

Acts like a molecular sieve

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

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

2. Gel Electrophoresis: 2. 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

2. Gel Electrophoresis: 2. Gel Electrophoresis: Attraction MigrationAttraction Migration

Negatively charged electrode at the end where wells are located

Positively charged electrode at opposite endNegatively charged DNA migrate towards

positive end due to attraction

2. Gel Electrophoresis: 2. Gel Electrophoresis: Rate of MigrationRate of Migration

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

Longer/larger DNA fragments migrate through gel slower

Rate of migration = 1/log(size)

Different DNA fragment lengths are 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

2. 2. Gel ElectrophoresisGel Electrophoresis: : Visualizing DNA FragmentsVisualizing DNA Fragments

Ethidium bromide is a fluorescent dye that makes DNA fragments visible by staining the gel

DNA fragments can then be isolated and purified

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

2. Gel Electrophoresis: 2. Gel Electrophoresis: Proteins too!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. Plasmids3. Plasmids

Small, circular double-stranded DNA that can enter and exit bacterial cells

Lack 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 and heavy metals

3. Plasmids3. Plasmids Foreign genes (ie. insulin) can be inserted into plasmids,

so bacteria can express gene and make its respective protein

Higher copy number of plasmids (number of individual plasmids) in bacteria– results in larger number of gene copies, thus more of its

respective protein is synthesized

3. Plasmids3. Plasmids 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

4. Transformation4. TransformationIntroduction of foreign DNA (usually a

plasmid) into a bacterium

Plasmids can be used as a vector (vehicle that DNA can be introduced to host cells) to carry a specific gene into a host cell

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

4. Transformation: 4. 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

4. Transformation: 4. Transformation: CompetenceCompetence