Biotechnology

Assessment statements

4.4.1 – 4.4.13

4.4.1 Polymerase Chain Reaction

• A technique that amplifies (clones) a small amount of DNA into millions of copies.

• Requires a very small initial sample and a few hours time

The Materials

• Target DNA (your sample you want to amplify)

• Taq polymerase (DNA polymerase isolated from a bacteria, Thermus aquaticus in a thermal spring in Yellowstone)

• DNA primers

• DNA nucleotides

• Taq polymerase remains active despite repeated heating and cooling.

• The ingredients are added to a test tube and placed into a thermal cycler that uses a computer to control the repetitive temperature changes required for PCR

The Methods

1. Melting – the mixture is heated to 94-96 oC for a minute or so to denature DNA into single strands

2. Annealing – the mixture is cooled to 50-65 oC and primers attach (by H bonds) to their complementary sequences on either side of the target sequence

3. Elongation – the mixture is brought to 72 oC for several minutes during which polymerase binds and extends a DNA complement from each primer

A video

• http://faculty.plattsburgh.edu/donald.slish/PCRmov.html

• After amplification the products of PCR are loaded into wells of an agarose gel and electrophoresed.

Gel Electrophoresis

• DNA is chopped into fragments using restriction enzymes (more on this later)

• Fragments are loaded in a gel that has small wells at one end.

• The gel is placed in a box that can be connected to an electric current

• The wells with the DNA are loaded at the negative end of the box and the current is turned on

• Because the phosphate groups on DNA are negatively charged the DNA migrates through the gel toward the positive pole

• The gel acts as a molecular sieve, separating the pieces of DNA by size.

An animation

• http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

A simulation

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

Sample Results

Restriction Enzymes

• Restriction enzymes (endonucleases) cut DNA at locations called palindromic sequences

• These cuts may result in either blunt or sticky ends

• Different DNA cut with the same restriction enzyme can be spliced together because they have the same sticky ends! This makes genetic engineering possible.

DNA Cloning

• Cloning employs plasmids, small circles of DNA found in prokaryotic cells that are supplemental to the bacterial cells main DNA

• Plasmids are removed from host cells and cut with restriction enzymes. The gene to be copied is mixed with the cut plasmids and complimentary ends align. DNA ligase joins them together. The plasmid is now referred to as a recombinant and can be used as a vector.

• The vector is inserted in a host bacterium and it is placed in a bioreactor.

• The host reproduces and copies the gene

• The host also expresses the gene and synthesizes the protein that the gene codes for.

• Bacteria can be created to make human proteins. Ex.) insulin

DNA Profiling

• Matching an unknown sample of DNA to a known sample. AKA DNA fingerprinting

• Utilizes Satellite (VNTR-variable number of tandem repeats) DNA - highly repetitive sequences of DNA from the non coding region of the genome.

• Different individuals have a unique length to their satellite regions that can be used to differentiate between individuals.

An article on DNA profiling

• http://learn.genetics.utah.edu/content/labs/gel/forensics/

A Simulation http://www.biotechnologyonline.gov.au/popups/int_dnaprofiling.html

Uses

• Crimes

• Paternity

• Selective Breeding – endangered species

• Animal migration

• Establishing evolutionary relationships

Click 4 biology

• http://click4biology.info/c4b/4/gene4.4.htm#four

• Check out this link for application of this technology and to see real gels!

Human Genome Project

• After watching “Cracking the Code of Life”, what ethical issues regarding the human genome concern you?

• http://www.pbs.org/wgbh/nova/genome/

Exploration

• At the website complete the following activities:– Take the survey. Submit your answer to see

how others voted on each issue.– Manipulating genes (read both parts 1 and 2)– Understanding heredity (make a list of the

people and each ones major contribution)– Explore a stretch of code (define hitchhiking

code, ancient code, sites of variation)– Sequence for yourself (complete the activity)

On your own time

• Nature vs. Nurture

• Journey into DNA

• Meet the decoders

Genetically Modified Organisms

• GMO’s have had an artificial genetic change using genetic engineering techniques such as gene transfer or recombinant DNA

Transgenic Plants

• An undesirable gene is removed and sometimes a desirable gene is put in its place.

• Scientists can create “designer plants” that exhibit greater productivity, resistance to pests, higher nutrient content etc…

• See Click 4 biology for examples and links! • http://click4biology.info/c4b/4/gene4.4.htm#nine

Transgenic Animals

• New genes can be inserted into animals so that they produce products that humans need.

• Ex.) sheep/cows may produce milk that contains clotting factor for those with hemophilia.

Pros and Cons of Genetically Modified Organisms

• Pros – more nutritious, productive, resistant crops; ability to produce rare proteins for medicines or vaccines; faster than selective breeding at giving desired traits

• Cons – genetically modified crops may pollinate wild species; crops may harm humans through toxicity or by causing allergy; may lead to a decline in biodiversity as engineered organisms increase in number.

Info on GMO’s

• http://www.csa.com/discoveryguides/gmfood/overview.php Mainly deals with crops.

• http://www.actionbioscience.org/biotech/sakko.html More pros and cons

• http://www.who.int/foodsafety/publications/biotech/20questions/en/ World Health Organization (available in several languages!)

Cloning

• A Clone is a group of genetically identical organisms or group of cells artificially derived from a single parent.

Dolly! Why she was unique!

• Dolly was the first clone whose genetic information did not originate from an egg!– An udder cell was collected and cultured. A cultured

cell was selected and its nucleus was removed.– An unfertilized egg was collected and its nucleus was

removed.– The egg and the somatic nucleus were fused.– The new cell began embryonic development and was

implanted in a surrogate mother– Dolly was born! A clone of the udder cell donor!

Therapeutic Cloning

• Uses undifferentiated cells, often derived from embryos

• http://www.dnalc.org/resources/animations/stemcells.html

Stem Cell Webquest

• http://outreach.mcb.harvard.edu/teachers/Summer07/JimDixon/SC_Biology_webquest.doc

Homework

• Page 110, #14-17