Genetic engineering and BiotechnologyTopic 4.4Or 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.

PCRPOLYMERASE CHAIN REACTIONUsed in researchStudy a particular sequence

Need of identical copiesmethod of cloninglots of time and work

It uses enzymes to replicate DNAEnzyme is isolated from bacteria and yeast.KARY MULLIS-developed in 1983Nobel prize -1993

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

Principle of PCRDesired DNA is heated---break the Hydrogen bondTwo strand separatePrimes are added to start ReplicationMixture is cooled. Primers bind to the original to ssDNA.Nucleotides and thermostable DNA- Polymerase is addedNucleotides will bond with exposed bases of the ssDNAThe original strand has formed a new CDNA.Cycle is repeated

Gel ElectrophoresisGel electrophoresis is a technique used to separate fragments of DNA.

Separates fragments as a function of size and charge.

Most types use Agarose to separate fragments.

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

Electrophoresis Technique An agarose gel is casted with several holes called wells at one end.The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution.Samples of digested DNA are placed in the wellsElectrical leads are attached to the ends of the box creating an electrical potential across the apparatus.Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus.Also, since the smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA.The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.

Finished Gel

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

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

DNA Fingerprinting ContdThis will therefore produce a unique banding pattern following a gel electrophoresis.

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

Crime sceneSmall amounts of DNA Amplify the DNA-PCRTwo strands are separatedRestriction enzymes- endonucleases used to cutSections will differ in size and chargeSeparated by Gel electrophoresisPattern of stripes and bands determined by the sequence of the bases

DNA Fingerprinting

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, semenc. Using relatives DNA to determine the identity of a victim-to determine the identity of the remains of dead people.

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 RomanovsDNA patterns could be established Conclusion-Bodies were likely to be the Romanov family

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

Human genome projectcommitment 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

Suggested in 19851990-started (3x109 base pairs) in human DNAIn 2003 the sequencing of the human DNA was 99.9% complete.Mapping of genes-listing and finding the locus of each human gene

Outcomes of having sequenced the entire human GenomeAn improved understanding of many genetic diseaseThe production of medicines (based on DNA sequences) to cure and/ or genetic engg. To remove the gene which causes the diseasesTo determine fully which genetic diseases any individual is prone to ( genetic screening leading to preventive medicine)

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

Genetic engineeringDeliberate manipulatipn of genetic material.This is possible due to-Universal genetic codeTransfer the genetic material from one species to anotherIntroduce human gene for making insulin into a bacteriumThe bacterium produces human protein insulin.

Gene transferRequires following elementsA vector-Plasmid-to carry the gene into the hostA host cellRestriction enzymesDNA ligase

Restriction EnzymesRestriction enzymes are compounds first isolated in the 1970'sThey function by selectively cutting DNA at specific sequences

Restriction EnzymesThese cuts usually occur in the following forms.The cut can be made straight across a base-pair sequence resulting in a "Blunt EndThe cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends"Blunt EndSticky end

Gene SplicingThe presence of sticky ends allows segments of DNA to be joined together. Since DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

Recombinant DNA

Gene Splicing contd..This allows for genes to be "cut & pasted" between organisms. This can be seen with production of human insulin.The DNA sequence of insulin is identified and cut out using a restriction enzyme.A plasmid from E. coli is removed and cut open using the same restriction enzymeSince both fragments have complimentary sticky ends and the gene for human insulin is integrated into the plasmidThe plasmid is then reinserted into a bacterial cell. This cell will produce insulin and is cultured. Human insulin can now be extracted and provided to diabetics.

Two examples-GM crops and animalsGMO-Genetically manipulated organismsCalled transgenic organisms

Flavr Savr1994-first GM food was sold commercially.Tomato was altered---stay fresh longerGene was introduced to block the enzyme which cause rottingNo longer available

Bt corn

G M Maize.A gene from Bacillus thuringiensis (Bt)Incorporated into maizePlants produce a toxin that makes them resistant to insectsBt crops are grown in US

Bt CornEuropean corn borer, ECB found also in USThe 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.

benefits of Bt cornThe 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.

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.

harmful effects of bt cornWill 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 timeResistant insects also make Bt spray useless as insecticide (Bt spray is considered to be relatively safe for humans and the environment).

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.

examplesGM mice--- to study the disease polioPossible treatment and prevention.Golden RiceRice is major part of their dietSuffer from Vit A deficiency---lead to blindnessRice store Kit A in leaves but not in rice grainsBy adding genes from daffodills and from bacteriumPlant stores a precursor of Vit A in the grains---yellow color

New kind of rice is now produced

One gene from maize and bacterium20 times more of beta carotene compared of Golden Rice. valuable source of Vit A opposed by environmentalist, and anti-globalisationists.

Cloning

cloneGroup of genetically identical organisms or group of cells derived from a single cellUsing differentiated cells is mostly somatic cells nuclear transfer-SCNT.

Reproductive cloning

Therapeutic cloning

Cloning: What it is Cloning is the process of making a genetically identical organism through nonsexual means. It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Since Dolly, several scientists have cloned other animals, including cows and mice. The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans Dolly, the first mammal clone

Dolly: A Mammal CloneDolly In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal. Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division. The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.

Clone a MIMI mousehttp://learn.genetics.utah.edu/content/tech/cloning/

Reproductive cloningCreates a new cloningDolly the sheepDolly 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.egEg., mare and her cloned foal

Therapeutic cloningInvolves stem cell researchHuman embryos are produced and allowed to grow for few days into small of ball of cellsThese 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.

Aims for cell therapyUsed Parkinsons diseaseBone marrow transplants, skin cells for burn victimsGrow new corneas

Ethical issues of Therapeutic cloning in humansArguments 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.

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.

***************