Biotechnology4.4 Syllabus points

4.4.1 PCR4.4.1 PCR Outline use of PCR to copy and amplify

minute quantities of DNA

PCR = polymerase chain reaction

Helps to get enough DNA to analyze when a sample is very small

Thermocycler - machine automates the copying of the DNA

Millions of copies in a few hours

PCRPCRUses DNA polymerase from bacteria that lives

in hot springs Thermus aquaticus

3 steps

Denature – heat up breaks double into single strand (about 98 degrees C)

Anneal – cool and primers can attach to either a top or bottom strand

Extension – Taq polymerase makes complementary strand

PCRPCRRepeated cycles of heating and cooling

stimulates the primers to attach to copies and copies of copies

Result is exponential increase in number of copies of DNA

4.4.24.4.2 Gel electrophoresis separates DNA by size

as move in electric field

DNA negative charge move towards positive pole

Agarose gel forms mesh of cross linked polymers

DNA stained to visualize the separated bands

4.4.34.4.3 Gel electrophoresis of DNA used in DNA

profiling

Matching an unknown sample with a known to see if they match up is DNA profiling

Identical band patterns means that is the individual in question – similar patterns usually mean the individuals are relatives

4.4.4 Applications of 4.4.4 Applications of DNA profilingDNA profiling

Paternity – legal identification of biological father

Forensic investigations – compare samples from crime scene with suspect

Cold cases reopen with new DNA technology such as PCR

Release of wrongly imprisoned individuals

4.4.5 Analyze gels4.4.5 Analyze gels Use gel banding patterns to determine

matches between individuals to solve crimes or determine paternity

4.4.6 Human Genome 4.4.6 Human Genome ProjectProject

Outline three outcomes of sequencing the complete human genome

1990-2003 international effort

Government and private labs

Aimed to identify the entire sequence of bases in human DNA

Created in essence a map of all genes on all 23 chromosomes

4.4.6 Human genome4.4.6 Human genome Allows identification of location of genes

causing diseases

Production of new medications by identifying products made by healthy individuals determine the gene responsible copy the gene and use it to produce the desired molecule

Evolutionary relationships and migration patterns of humans

4.4.7 Gene transfer4.4.7 Gene transfer Genes can be transferred between species

because DNA is universal language and code for the same amino acids

So gene will produce the same protein no matter what organism it is placed into

Examples gene Examples gene transfertransfer

Cold resistant tomatoes

Bt-corn (Bacillus thuringiensis)

Spider silk goats

4.4.8 Technique Gene 4.4.8 Technique Gene TransferTransfer

Cut and paste genes using restriction enzymes AKA endonucleases find and cut at target sequences

Paste genes using DNA ligase enzyme which recognizes the sticky ends of the fragments and attaches them

Copy paste using Copy paste using plasmid DNAplasmid DNA

Use host cell often Escherichia coli or yeast

Prokaryotes most DNA in single chromosome but also have extra DNA is small loops called plasmids

To copy DNA it needs to be pasted into a plasmid

4.4.8 Pasting into 4.4.8 Pasting into plasmid plasmid

Remove plasmid from cytoplasm of bacteria

Cut open plasmid using restriction enzyme

Paste new gene using DNA ligase into the open plasmid

Modified plasmid called a recombinant plasmid

Recombinant plasmid used to deliver new gene into genome of target organism

4.4.9 Genetically 4.4.9 Genetically Modified organismsModified organisms

Golden rice – beta carotene into rice prevent blindness

Salt resistant tomatoes

Proponents – will help solve world hunger

Critics – world hunger is distribution not production issue

4.4.10 Benefits/Risks 4.4.10 Benefits/Risks GMOGMO

Benefits:

• Improve food production

• Reduce chemicals if plants have own pest control substances

• Reduce cost and pollution of making medicines

• Farmers have added control of crops and livestock

• Globally GM crops need less water and pesticides

4.4.10 Risks4.4.10 RisksRisks- long term risks unknown

• Pollen escapes into wild integrated possibly into wild genome

• Genes could cross species – seen in labs could it happen in wild?

• Bt crops harmful to humans – pesticide throughout plant not just on surface

• Allergies

• Large corporations will own large part of food supply

• High tech not always better than simple solutions

• Decrease in biodiversity

4.4.11 Cloning4.4.11 Cloning Define clone

Group of identically identical organisms or group of cells artificially derived from a single parent

Farmers have cloned plants for years by regenerating plant material

4.4.12 Technique 4.4.12 Technique cloningcloning

Enucleate unfertilized egg and transfer nuclear material from donor cell

Electrical current fuses egg and nucleus

Zygote divides and forms embryo which is implanted in surrogate mother

Donor cell may be adult differentiated cell (non gamete) rather than from an egg cell

Offspring is clone of donor organism

4.4.13 Ethics 4.4.13 Ethics therapeutic cloningtherapeutic cloning

Cloning using undifferentiated cells

AKA embryonic stem cells

Requires production of human embryos

Is it ethical to generate new humans for medical research?

However, stem cells allow repair of burns, new heart muscle, new kidney tissue

Types of CloningTypes of Cloning Therapeutic cloning makes new tissues

Reproductive cloning makes new organisms