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Genetic engineering and Biotechnology Genetic engineering and Biotechnology Topic 4.4 Topic 4.4 Or how I stopped Or how I stopped worrying and learned to worrying and learned to love the sheep.” love the sheep.”

Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

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Page 1: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Genetic engineering and BiotechnologyGenetic engineering and BiotechnologyTopic 4.4Topic 4.4

““Or how I stopped worrying and Or how I stopped worrying and learned to love the sheep.”learned to love the sheep.”

Page 2: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”
Page 3: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

polymerase chain reaction (PCR)

• scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

• The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Page 4: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

PCR

• POLYMERASE CHAIN REACTION

• Used in research

• Study a particular sequence

• Need of identical copies

• method of cloning—lots of time and work

Page 5: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• It uses enzymes to replicate DNA

• Enzyme is isolated from bacteria and yeast.

• KARY MULLIS-developed in 1983

• Nobel prize -1993

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

Page 6: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Principle of PCR• Desired DNA is heated---break the Hydrogen bond• Two strand separate• Primes are added to start Replication• Mixture is cooled. • Primers bind to the original to ssDNA.• Nucleotides and thermostable DNA- Polymerase is

added• Nucleotides will bond with exposed bases of the

ssDNA• The original strand has formed a new CDNA.

• Cycle is repeated

Page 7: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”
Page 8: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Gel ElectrophoresisGel Electrophoresis• Gel electrophoresis is a Gel electrophoresis is a

technique used to separate technique used to separate fragments of DNA.fragments of DNA.

• Separates fragments as a Separates fragments as a function of size and charge.function of size and charge.

• Most types use Most types use AgaroseAgarose to to separate fragments.separate fragments.

• AgaroseAgarose is a porous gel. It is a porous gel. It can allow the passage of can allow the passage of molecules through, molecules through, however, larger molecules however, larger molecules move more slowly through move more slowly through it since they cannot it since they cannot squeeze through the pores squeeze through the pores as easily as smaller as easily as smaller molecules.molecules.

Electrophoresis ApparatusElectrophoresis Apparatus

Page 9: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Electrophoresis TechniqueElectrophoresis Technique • An agarose gel is casted with several An agarose gel is casted with several

holes called wells at one end.holes called wells at one end.• The gel is placed in an electrophoresis The gel is placed in an electrophoresis

box which is filled with an electrolyte box which is filled with an electrolyte buffer solution.buffer solution.

• Samples of digested DNA are placed Samples of digested DNA are placed in the wellsin the wells

• Electrical leads are attached to the Electrical leads are attached to the ends of the box creating an electrical ends of the box creating an electrical potential across the apparatus.potential across the apparatus.

• Because DNA has a negative electrical Because DNA has a negative electrical charge. It is "pulled" towards the charge. It is "pulled" towards the positive side of the apparatus.positive side of the apparatus.

• Also, since the smaller molecules Also, since the smaller molecules travel faster through the agarose. travel faster through the agarose. Over time this separates the various Over time this separates the various sized fragments of DNA.sized fragments of DNA.

• The gel is then removed and stained The gel is then removed and stained for DNA. This results in a gel which for DNA. This results in a gel which shows several bands of stained DNA.shows several bands of stained DNA.

Page 10: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Finished GelFinished Gel

Page 11: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

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

Page 12: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

DNA FingerprintingDNA FingerprintingDNA profiling is also known as DNA fingerprintingDNA profiling is also known as DNA fingerprintingDNA is now a powerful tool in identification.DNA is now a powerful tool in identification.Based on the fact that the amount of "junk DNA" differs uniquely between Based on the fact that the amount of "junk DNA" differs uniquely between individuals.individuals.Structural genes are often separated by large regions of repeating base Structural genes are often separated by large regions of repeating base pairs.pairs.The number of these repeats is unique to an individual.The number of these repeats is unique to an individual.Therefore when DNA from a person is cut with a restriction enzyme, the Therefore when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual.length of the fragments will be unique to an individual.

Page 13: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

DNA Fingerprinting Contd…DNA Fingerprinting Contd…• This will therefore This will therefore

produce a unique produce a unique banding pattern banding pattern following a gel following a gel electrophoresis.electrophoresis.

• This test is highly This test is highly accurate, and the accurate, and the probability of another probability of another individual possessing individual possessing an identical banding an identical banding pattern is estimated as pattern is estimated as around around 1:14,000,000,000. 1:14,000,000,000.

Page 14: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Crime scene

• Small amounts of DNA

• Amplify the DNA-PCR

• Two strands are separated

• Restriction enzymes- endonucleases used to cut

• Sections will differ in size and charge

• Separated by Gel electrophoresis

• Pattern of stripes and bands determined by the sequence of the bases

Page 15: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

DNA FingerprintingDNA Fingerprinting

Page 16: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Applicationa. Paternity profiling

-each band shown on the DNA Profile of a child must correspond with a band of the father or the mother.

b. Forensic investigation-

-Compare DNA from the suspect with DNA from the crime scene sample (blood, hairs, semen

c. Using relative’s DNA to determine the identity of a victim

-to determine the identity of the remains of dead people.

Page 17: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• For Eg., Tsar of Russia and his family was shot during the Russian revolution and bodies were shown to prove it.

• By taking blood samples of distant relatives of the Romanovs

• DNA patterns could be established

Conclusion-

Bodies were likely to be the Romanov family

Page 18: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

see

• http://en.wikipedia.org/wiki/Genetic_ fingerprinting ------for some ideas of problems when using DNA profiling as evidence.

Page 19: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Human genome project

• commitment undertaken by the scientific community across the world.

• International Human Genome Organisation –is an excellent example of how collaboration of scientists across the world can benefit all of us.

To determine the locations and structure of all genes in the human chromosomes

Data was pooled

Page 20: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• Suggested in 1985

• 1990-started (3x109 base pairs) in human DNA

• In 2003 the sequencing of the human DNA was 99.9% complete.

• Mapping of genes-listing and finding the locus of each human gene

Page 21: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Outcomes of having sequenced the entire human Genome

• An improved understanding of many genetic disease

• The production of medicines (based on DNA sequences) to cure and/ or genetic engg. To remove the gene which causes the diseases

• To determine fully which genetic diseases any individual is prone to ( genetic screening leading to preventive medicine)

Page 22: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• Research into a particular disease can focus on only gene(s)

• Provide more info about evolutionary paths by comparing similarities and differences in genes between species.

• Info is valuable BUT it could be abused.—insurance companies, prospective employer

and society faces the challenge of coming to terms with the ethical issues

Page 23: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Genetic engineering

• Deliberate manipulatipn of genetic material.

• This is possible due to-

• Universal genetic code

• Transfer the genetic material from one species to another

• Introduce human gene for making insulin into a bacterium

• The bacterium produces human protein insulin.

Page 24: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Gene transfer• Requires following elements

• A vector-Plasmid-to carry the gene into the host

• A host cell

• Restriction enzymes

• DNA ligase

Page 25: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Restriction EnzymesRestriction Enzymes

• Restriction enzymes are compounds first Restriction enzymes are compounds first isolated in the 1970'sisolated in the 1970's

• They function by selectively cutting DNA at They function by selectively cutting DNA at specific sequences specific sequences

Page 26: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Restriction EnzymesRestriction Enzymes

• These cuts usually occur These cuts usually occur in the following forms.in the following forms.

• The cut can be made The cut can be made straight across a base-straight across a base-pair sequence resulting in pair sequence resulting in a "a "Blunt EndBlunt End““

• The cut can be made in The cut can be made in an offset manner leaving an offset manner leaving exposed nucleotide exposed nucleotide sequences. These sequences. These exposed sequences are exposed sequences are called "called "Sticky EndsSticky Ends""

Blunt EndBlunt End

Sticky endSticky end

Page 27: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Gene SplicingGene Splicing• The presence of The presence of

sticky ends allows sticky ends allows segments of DNA to segments of DNA to be joined together.be joined together.

• Since DNA strands Since DNA strands which have been cut which have been cut by the same by the same restriction enzyme restriction enzyme can easily bond can easily bond together according together according to base pairing to base pairing rules. rules.

Page 28: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Recombinant DNA

Page 29: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”
Page 30: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Gene Splicing contd..Gene Splicing contd..• This allows for genes to be This allows for genes to be

"cut & pasted" between "cut & pasted" between organisms. This can be seen organisms. This can be seen with production of human with production of human insulin.insulin.

• The DNA sequence of insulin The DNA sequence of insulin is identified and cut out using a is identified and cut out using a restriction enzyme.restriction enzyme.

• A plasmid from A plasmid from E. coliE. coli is is removed and cut open using removed and cut open using the same restriction enzymethe same restriction enzyme

• Since both fragments have Since both fragments have complimentary sticky ends and complimentary sticky ends and the gene for human insulin is the gene for human insulin is integrated into the plasmidintegrated into the plasmid

• The plasmid is then reinserted The plasmid is then reinserted into a bacterial cell. This cell into a bacterial cell. This cell will produce insulin and is will produce insulin and is cultured. Human insulin can cultured. Human insulin can now be extracted and provided now be extracted and provided to diabetics.to diabetics.

Page 31: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Two examples-GM crops and animals

• GMO-Genetically manipulated organisms

• Called transgenic organisms

Page 32: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Flavr Savr• 1994-first GM food was sold commercially.

• Tomato was altered---stay fresh longer

• Gene was introduced to block the enzyme which cause rotting

• No longer available

Page 33: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Bt corn

• G M Maize.

• A gene from Bacillus thuringiensis (Bt)

• Incorporated into maize

• Plants produce a toxin that makes them resistant to insects

• Bt crops are grown in US

Page 34: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”
Page 35: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Bt Corn

• European corn borer, ECB found also in US

• The ECB through stems and leaves of the corn plant and will damage vascular bundles

• disrupt the transport of water and nutrients through the plant.

• It can also weaken the stems and leaves so that the plant or leaves may break

• Bt corn is already in commercial use.

Page 36: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

benefits of Bt corn

• The damage caused by the ECB is much reduced.

• Bt corn is slightly more expensive, but the difference is less than one extra application of insecticide.

• Non-Bt corn needs to be checked often for signs of ECB - less checking needed for Bt corn.

Page 37: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• Less insecticide needed means less impact on the environment and lower health risks for the worker(s)

•Seems to reduce the infection with fungus so mycotoxin (poisons produced by fungi) levels are lowered.

Mycotoxins are difficult to remove by cooking/freezing and may go into the food chain and be found in meat of animals which ate the infected corn. Mycotoxins can be a hazard to human and animal health.

Page 38: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

harmful effects of bt corn• Will also kill some other insects (though

many are not affected).

• Insects may develop resistance to Bt toxin because they are exposed to it all the time

• Resistant insects also make Bt spray useless as insecticide (Bt spray is considered to be relatively safe for humans and the environment).

Page 39: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• It is difficult to prevent pollen (with the Bt gene) from travelling outside the field where the Bt corn is grown

-it may fertilise non-Bt corn e.g.

organically grown corn which can then

no longer be sold as organic corn.

-it may fertilise wild relatives and make

them more resistant to insects and have

them dominate the niche they live in.

-This would result in loss of biodiversity.

Page 40: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

examples• GM mice--- to study the disease polio

• Possible treatment and prevention.

• Golden Rice

• Rice is major part of their diet

• Suffer from Vit A deficiency---lead to blindness

• Rice store Kit A in leaves but not in rice grains

• By adding genes from daffodills and from bacterium

• Plant stores a precursor of Vit A in the grains---yellow color

Page 41: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• New kind of rice is now produced

• One gene from maize and bacterium

• 20 times more of beta carotene compared of Golden Rice.

• valuable source of Vit A

• opposed by environmentalist, and anti-globalisationists.

Page 42: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

CloningCloning

Page 43: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

clone• Group of genetically identical organisms or

group of cells derived from a single cell

• Using differentiated cells is mostly somatic cells nuclear transfer-SCNT.

• Reproductive cloning

• Therapeutic cloning

Page 44: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Cloning: What it isCloning: What it is• CloningCloning is the process of making a is the process of making a

genetically identical organism through genetically identical organism through nonsexual means.nonsexual means.

• It has been used for many years to It has been used for many years to produce plants (even growing a plant produce plants (even growing a plant from a cutting is a type of cloning).from a cutting is a type of cloning).

• Animal cloning has been the subject Animal cloning has been the subject of scientific experiments for years, of scientific experiments for years, but garnered little attention until the but garnered little attention until the birth of the first cloned mammal in birth of the first cloned mammal in 1997, a sheep named 1997, a sheep named DollyDolly. .

• Since Dolly, several scientists have Since Dolly, several scientists have cloned other animals, including cows cloned other animals, including cows and mice. and mice.

• The recent success in cloning The recent success in cloning animals has sparked fierce debates animals has sparked fierce debates among scientists, politicians and the among scientists, politicians and the general public about the use and general public about the use and morality of cloning plants, animals morality of cloning plants, animals and possibly humans and possibly humans Dolly, the first mammal cloneDolly, the first mammal clone

Page 45: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Dolly: A Mammal CloneDolly: A Mammal Clone

• DollyDolly

• In 1997, cloning was revolutionized In 1997, cloning was revolutionized when when Ian WilmutIan Wilmut and his colleagues at and his colleagues at the Roslin Institute in Edinburgh, the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep Scotland, successfully cloned a sheep named named DollyDolly. Dolly was the first cloned . Dolly was the first cloned mammal. mammal.

• Wilmut and his colleagues transplanted Wilmut and his colleagues transplanted a nucleus from a mammary gland cell a nucleus from a mammary gland cell of a Finn Dorsett sheep into the of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface enucleated egg of a Scottish blackface ewe. ewe.

• The nucleus-egg combination was The nucleus-egg combination was stimulated with stimulated with electricityelectricity to fuse the to fuse the two and to stimulate cell division. two and to stimulate cell division.

• The new cell divided and was placed in The new cell divided and was placed in the uterus of a blackface ewe to the uterus of a blackface ewe to develop. Dolly was born months later. develop. Dolly was born months later.

Page 46: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Clone a MIMI mouse

• http://learn.genetics.utah.edu/content/tech/cloning/

Page 47: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Reproductive cloning• Creates a new cloning

• Dolly the sheep

• Dolly is known as SCNT

• it is theoretically possible to apply the same technique to cloning other species.

• Horses are an example of a species cloned successfully, but attempts with several other species have been less successfull.eg

• Eg., mare and her cloned foal

Page 48: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Therapeutic cloning• Involves stem cell research

• Human embryos are produced and allowed to grow for few days into small of ball of cells

• These cells are not specialised but when SCNT is used the cells can grow into any different specialised tissues.

• Other sources of stem cells from umbilical cord or cells from aborted fetuses.

Page 49: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

• Aims for cell therapy

• Used –Parkinson’s disease

• Bone marrow transplants, skin cells for burn victims

• Grow new corneas

Page 50: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Ethical issues of Therapeutic cloning in humans

• Arguments in favor of therapeutic cloning focus on:

• the ability to cure serious diseases with cell therapy:

• currently leukemia and

• in the future possibly cancer and diabetes.

Page 51: Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

Some of the concerns raised about therapeutic cloning relate

• fears of it leading to reproductive cloning

• use of embryonic stem cells involves the creation and destruction of human embryos (although it is possible to use embryos left over from IVF treatment which would be destroyed anyway)

• embryonic stem cells are capable of many divisions and may turn into tumors.

• Ethical aspects of cloning are difficult to discuss since a lot of the benefits are currently not yet realised. They are potential benefits.