DNA& Biotechnology

I. What is DNA?• Deoxyribonucleic acid• Polymer• Made of long chains of nucleotides

– Phosphate group, sugar, & a nitrogen base• 4 bases: C, G, A, T

– C G, G C– A T, T A– Bases held together by hydrogen bonds

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What is the structure of DNA?• 2 strands running opposite each other

– Sides are alternating deoxyribose sugar & a phosphate molecule

- N-bases are connected to the sugar

• Form a twisting ladder, double helix

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DNA’s job• DNA is transcribed into RNA which codes for

amino acids and makes proteins• Not all DNA codes for proteins• Coding sequences = exons• Noncoding parts = introns • RNA splicing trims out introns

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p. 14-16 of NG, 30-1

Where is DNA found?

• Coiled up –making chromosomes– Found inside the nucleus of cells

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p.6-7 NG

Biotechnology

• The study & manipulation of living things or their component molecules, cells, etc.

biologycorner.com

Early biotech

• Planting crops• Breeding animals to get specific traits• Using yeast to turn

– Fruit juice into wine– Malt and hops into beer– Making bread rise

• Using bacteria to turn milk into yogurt and cheese

From Amanda Noller via Pamela Peters, from Biotechnology: A Guide To Genetic Engineering. Wm. C. Brown Publishers, Inc., 1993.

Current uses of biotech• Amplifying DNA to see it• Sequencing genomes• Gene splicing• Recombinant DNA/ Transformation• Creating effective medicines• Genetic Engineering• Cloning• Stem Cell Research• And so much more…• http://www.iptv.org/exploremore/ge/what/insulin.cfm

From Amanda Noller via Pamela Peters, from Biotechnology: A Guide To Genetic Engineering. Wm. C. Brown Publishers, Inc., 1993

Isolating & manipulating DNA• Why?• Changing DNA nucleotides can alter proteins

– Making medicines/hormones ex: insulin p.64– http://www.youtube.com/watch?v=tJP_6nAPES4

chemicalconnections.org

Chromatography

• a set of techniques used to separate different compounds.

• Uses: isolating new compounds, analyzing subtle differences between different samples, and even in the sequencing of DNA.

Paper chromatography

• stationary phase (a solid, or a liquid supported on a solid) • mobile phase (a liquid or a gas).• The mobile phase flows through the stationary phase and

carries the components of the mixture with it.• Different components travel at different rates.• In paper chromatography, the stationary phase is a uniform

absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

Rf values• Some compounds in a mixture travel almost as far as the

solvent does; some stay much closer to the base line. • The distance travelled relative to the solvent is called the Rf

value. For each compound it can be worked out using the formula:

• Rf = distance traveled by compound

distance traveled by solvent• For example, if one component of a mixture travelled 9.6 cm

from the base line while the solvent had travelled 12.0 cm, then the Rf value for that component is: 9.6/12 = 0.8

Gel electrophoresis

• Separation technique• Visual way to see the pieces of DNA• Uses electrical impulses to pull the DNA

through a matrix like jello (called Agarose)• DNA in negatively charged, so it moves

towards a positive pole and away from the negative

• Separated by size, shape, & charge• http://www.dnalc.org/resources/animations/gelelectrophoresis.html

• The cut DNA is mixed with blue dye and loaded into a gel

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• DNA moves towards positive end (due to the negative charges of the phosphates)

• The dye moves faster than the smallest DNA fragments

• Smaller the fragments the faster they move

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Using a micropipetter

• http://www.youtube.com/watch?v=uEy_NGDfo_8 • Set desired volume by turning the dial• Max volume is on top of pipette• Put sterile tip on from box• To load sample, press down until 1st stop• Release button to draw up sample• To release sample into container, push down on button, past

the 1st stop• Withdraw pipette from container before releasing the button

PCR

• Polymerase chain reaction• Used for forensics, medicine, research, and

more• Amplifies small sections of DNA to get LOTS of

DNA

PCR8

What you need

• DNA - you need something to copy!• Taq polymerase - copies the DNA• Primers - get the polymerase going• dNTP - the As, Ts, Cs, and Gs• buffered (pH controlled) tube of water• thermocycler

thermocycler

Taq polymerase

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In the thermocycler…1. Heat to 95ºC (Denature)• DNA denatures into its two strands

2. Cool down to 58ºC- 65°C (Annealing)• Primer sequences stick to the template

3. Heat to 72ºC (Synthesis)• Taq polymerase attach at the primer and make a

complimentary copy of the DNA

4. Repeat

• Start with one copy– One copy, one cycle- 2 copies– 2 copies, one cycle- 4 copies– 4 copies, one cycle- 8 copies

• Typical runs include between 25-35 cycles• Finish with billions of identical copies of the original

DNA fragment

• http://learn.genetics.utah.edu/content/labs/pcr/

Sources for pictures• 1. http://www.scq.ubc.ca/a-monks-flourishing-garden-the-basics-of-molecular-

biology-explained/• 2. • 3. http://biolibogy.com/chemistryoflife.html• 4.http://publications.nigms.nih.gov/thenewgenetics/chapter1.html • 5.http://publications.nigms.nih.gov/thenewgenetics/chapter1.html#dnastr• 6-9. Amanda Noller’s Biotechnology ppt• 12. http://andrew.cmu.edu• 13. http://coloradoindependent.com