DNA Technology

Biotechnology Tools & TechniquesRecombinant DNA – fragment of DNA composed of DNA sequences originating from at least two different sourcesRestriction Endonucleases – aka restriction enzymes– molecular scissors that cut double-stranded DNA at a specific base-pair sequence

Biotechnology Tools & Techniques

• Recognition Site- A specific sequence within double-stranded

DNA where restriction enzymes recognize and cut

- Usually palindromic (same front & back…eg. level, stats, racecar)

Biotechnology Tools & Techniques• Blunt ends- DNA fragment from a restriction enzyme

where the ends are base paired- Eg. SmaI, AluI• Sticky ends- DNA fragment from a restriction enzyme

where the ends contain short, single-stranded overhangs

- Eg. EcoRI, SalI, HindIII

Restriction Endonucleases• Isolated and purified solely from bacteria• Essentially a bacteria’s immune system• Also how a virus can break down host DNA• Named according to the bacteria from which

they originate. Example, in Escheria coli (E. Coli), the restriction enzyme is called EcoRI

• BAMHI comes from Bacillus amyloliquefaciens; H is the strain, 1 is b/c it’s the first endonuclease isolated from it

Restriction Endonuclease Cut Sites

Restriction Endonucleases• Recognize and bind to sequences which are

4 to 8 nucleotides long• Eg. EcoRI looks for 5’ GAATTC 3’

3’ CTTAAG 5’ and cleaves (cuts) between G and A• A 6 base-pair sequence like this would occur

every 4x4x4x4x4x4 = 46=4096 base pairs

Methylases• Specific enzymes found in prokaryotes &

eukaryotes• In prokaryotes, they modify the recognition site

(add a methyl group to one of the bases) which prevents the DNA from being cleaved

• When foreign DNA is inserted, it is not methylated, so the desired gene fragment is protected from being cleaved

• In eukaryotes, methylases control transcription

DNA Ligase• Enzyme used to join the cut strands of DNA

together (best for sticky ends)• T4 DNA ligase (from T4 bacteriophage virus)

works better for blunt ends

Gel Electrophoresis• Process of

separating DNA on the basis of size by sorting through a gel meshwork

• Like a molecular sieve

Gel electrophoresis• DNA is neg. charged due to phosphate group• DNA already subjected to restriction

enzymes (pieces of various sizes) are loaded in wells with a buffer solution containing glycerol (dye to view the DNA)

• Larger fragments move through the agarose gel more slowly, smaller fragments move faster and farther

Gel electrophoresis• Uses DC current to place a neg charge at the

near end (closest to DNA); pos charge at the far end

• Once complete, fragments are made visible by staining (often ethidium bromide which fluoresces under UV light

• Not just nucleic acids – can run proteins using polyacrylamide gels

Plasmids• Recall that bacteria contain a singular

circular chromosome, along with many small, circular pieces of DNA called plasmids

• Plasmids carry genes which confer antibiotic resistance, as well as resistance to toxic heavy metals and industrial chemicals

• We can use plasmids for biotechnology, since bacteria are able to express foreign genes inserted into plasmids

Plasmids• Relationship between bacteria and plasmids

is endosymbiotic – both benefit• Engineered plasmids contain a region that

can be cut by many restriction endonucleases and found nowhere else on the plasmid – called the multiple cloning site

• In which gel lane was this plasmid run?

Steps in Recombinant DNA1. Plasmid DNA is cut open using R.E.2. Gene fragment to be inserted is opened

using same R.E., therefore ends will match3. DNA ligase joins fragment to the plasmid4. Plasmid is introduced into bacterial cell via

transformation

Recombinant DNATransformation – introduction of foregin DNA, usually by a plasmid or virus, into a bacterial cellVectors – vehicles by which DNA is introduced into host cells (bacteria or viruses)Host cell – the cell that contains the foreign DNA and whose machinery is being used to express that DNA

Homework• Good summary chart on Pg. 289 - 290• Do Q 1-6, 8, 11, 14, 15, 16, 17

Polymerase Chain Reaction (PCR)

PCR• Amplification of DNA sequence by repeated cycles of

strand separation and replication• Proposed by Kary Mullis in 1987; awarded Nobel

Prize in Chemistry in 1992 for the discovery • Direct replication of a DNA sequence without

insertion into a plasmid

Steps in PCR

1. Heat DNA to 94-96°C to separate strands2. Two primers (chunks of single-stranded DNA) are

made which correspond to the beginning and end of DNA to be copied

3. Heat to 72°C to extend primers using Taq polymerase

4. Separate strands and anneal (join) primers5. Extend primers 6. Repeat steps 4-6 for 30 – 40 cycles

PCR• Useful in criminal investigations (forensics), medical

diagnoses, and genetic & evolutionary biology (common ancestry)

• Only need small amounts of DNA

Restriction Fragment Length Polymorphism (RFLP)

RFLP• Polymorphism – any difference in DNA sequence that

can be detected between individuals• Only identical twins would not be polymorphic• RFLP is a process whereby differences in DNA

fragment lengths between individuals are compared

Steps in RFLP

1. DNA extracted from sample and cut by R.E.’s2. Digested fragments are run on a gel, which is placed

in a denaturing solution against a nylon membrane 3. Single stranded DNA migrates from gel to nylon

membrane using electric current = “Southern blotting”

4. Membrane soaked in solution with radioactive probes

Steps in RFLP

5. Radioactive probes anneal at specific locations6. Membrane placed against X-ray film for 2 – 3 weeks7. Radioactive probes burn image in X-ray film8. Developed X-ray film called an autoradiogram

Homework• Summary charts Pg. 300 and 303

Medical Applications• Development of insulin by GE in the 1980’s• HIV can be detected by PCR soon after infection• Genetic mutations can be detected before the

manifestation of symptoms• Being able to pinpoint the location of genes will lead

to more developments in gene therapy (eg. Gene inserted into adrenal gland cells which secrete a transmitter which inhibits the sensation of pain)

Agricultural Applications• Transgenic plants – 1981 (Nestor and Chilton)

Ex. Flavr Savr Tomato (gene for ripening in fruit)• Crops have been genetically engineered to increase

yield, hardiness, uniformity, herbicide tolerance, and insect and virus tolerance

• Insert polyester gene into a cotton plant to make poly-cotton fibers

• Transgenic corn – produces Bt toxin which kills butterflies!

Forensic Applications• 1987 – first court case using RFLP where accused was

convicted• Valuable tool for investigators, but cannot convict on

its own• Both PCR and RFLP are used in criminal investigations

depending on state of the DNA sample• Both techniques provide a ‘fingerprint’