1.Genetics III Genetics and Biotechnology

2. What is biotechnology Teddy 3. What is biotechnology United Nations Convention on Biological Diversity "Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." 4. Biotechnology past and present 5. 6. Biotechnology

Genetics counseling and diagnosis

Gene therapy

Human genome project

Plant and animal cloning

Genetic engineering and GMO

DNA fingerprinting

7. Who Prof. Tsui Lap Chee Profession Vice-Chancellor of HKU Academic contributions? identified the defective gene, namelyCystic fibrosis transmembrane conductance regulator(CFTR), that causescystic fibrosis 8.

Cystic fibrosis

Cystic fibrosis affects 1 in every 3,000 births

Cause: an inherited autosomal recessive gene

Symptoms and effects:

Very sticky mucus produced in lungs and pancreas causing lung infections and inability to absorb food from the gut properly.

9. Types of genetic testing

Prenatalor post natal counselling

http://www.nsgc.org/

10.

Case study: genetics counselor

11. What is his chance of having offspring with CF diseases?(CF gene is a recessive allele.)

The man need to determine if he is a carrier.

• If he is a carrier

• • Ifher wife doesn't have family history of CF disease, the chance of their offspring having the disease is rather low.

• • Ifher wife has family history of CFdisease, she need to know whethershe is a carrier.

• • • If she is a carrier, the chance ofhaving a CF offspring is 25%.(Aa x Aa)

12. What test would you advice him to do in order to determine whether he is a carrier of the CF gene?

He should take a CF carrier test (a blood test) to determine if he gets the abnormal gene that causes CF.

13. How can genetic counseling or genetic diagnosing help them to get better prepared for the birth of the child?

They can make an informed decision about pregnancy.

undergo an invasive diagnostic procedure during pregnancy to determine whether their fetus is affected

choose pregnancy termination to prevent birth of an affected infant.

opt for preimplantation genetic diagnosis and the selective transfer of embryos without CF; but this requires in vitro fertilization

14.

2 .Diagnostic to make or confirm diagnosis, generally where person already exhibits signs or symptoms of a disorder

Genetic disorders that can be diagnosed: e.g. Thalassemia, diabetes, leukemia, breast cancer.....

15. 16. Gene therapy - definition

The specific treatment of genetic disease

Modifying the expression of a persons genes towards a therapeutic goal

17. 18. The methodology of gene therapy

Identification of defective gene

Genetic engineering using normal/functional gene

Transfer of this material into target cells (gene delivery)

Integration of DNA into target cell genome

Correction of defect

19. Contributions of gene therapy: Good news : Promising advances during the last two decades in recombinant DNA technology . Bad news : Efficacy in any gene therapy protocol not definitive. 1.Shortcomings in all current genetransfer vectors. 2.Inadequate understanding ofbiological interactions of vector andhost. 20. Contributions of gene therapy 1.Cancer

Enhancement of immunological responses to the tumour

Modification of anti-oncogenes

Manufacture of anti-cancer factors

https://sites.google.com/a/luther.edu/genetics/students/chris-nevala-plagemann

21. Contributions of gene therapy 2.Infectious Diseases:HIV

Direct interference with viral processes

Genetic vaccination with HIV proteins

22. 3.Inherited/monogenic disorders: Parkinsons disease 23. Case study

Treatment ofS evereC ombinedI mmuno D eficiency (SCID)

Genetic defects cause decreased T and B cells.

Affects 1-75,000 births.

Mostly males (most common form is X-linked)

Cause: ADA (adenine deaminase) deficiency

Treatment options

• Germfree environment

• Bone marrow transplant

• Gene therapy

24. 10-3-02 : France and US (FDA) halted SCID gene therapy due to leukemia-like side effects in one child.Not clear whether this is related to the gene therapy itself. 1/14/03:FDA suspended 30 gene therapy trials using retrovirus vectors due to another case of leukemia. clinical trials results :Detectable levels of T cells containing the introduced gene were found in the blood within 30 and 60 days, respectively, and their numbers increased progressively until normal levels were reached.After 3 months, the patients were also able to make antibodies in response to vaccination against diphtheria, tetanus, and pertussis. 25. 26. Which are the 3 biggest science projects in the history of mankind? 27. 1942-45 Manhattan project 28. 1967-72 Apollo project 29. 1990-2003 Human Genome Project 30. Human Genome Project

Project goals :

identifyall the approximately 30,000-35,000 genes in human DNA,

determinethe sequences of the 3 billion chemical base pairs that make up human DNA,

31. Human Genome Project

Milestones :

June 2000 completion of a working draft of the entire human genome

February 2001 analyses of the working draft are published inNature

2003 completed

32. Some results Take a look at human chromosome: http://www.ensembl.org/Homo_sapiens/ Home page of the human genome project http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml 33. Characteristics / number of genes The total number of genes is estimated at 30,000 to 35,000 much lower than previous estimates. Almost all (99.9%) nucleotide bases are exactly the same in all people. (Side track questions: any 2 chimpanzees are more dislike than any 2 human, why?) The functions are unknown for over 50% of discovered genes. 34. How It's Arranged?

Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231).

Genes appear to be concentrated in random areas along the genome, with vast expanses of noncoding DNA between.

Less than 2% of the genome codes for proteins.

Repeated sequences that do not code for proteins ("junk DNA") make up at least 50% of the human genome.

35. Applications

Over 30 genes have been pinpointed and associated with some diseases (e.g. breast cancer, muscle disease, deafness, and blindness).

Additionally, finding the DNA sequences underlying such common diseases provide focused targets for the development of effective new therapies.

In the past, researchers studied one or a few genes at a time. With whole-genome sequences known, they can approach questions systematically and on a grand scale.

36. Discussion Questions 1. If the genome study obtain a franchise,how can we protect the right of the poor? 2. Now, the main area of application of the genome study is for medical purpose. However, with the same technique, it is also possible to manipulate genes for other purposes, such as increasing the longevity of life and I.Q. If then, is it ethical to create such "perfect" human beings? 3. Is it ethical to change the genes of other plants and animals? What are the risks behind? 4. It may be possible to prepare a tailor-made health-care program for each individual according his or her genes.Will the privacy of individuals be respected? 37. Improving Plant and Animal Performance 38. Improvement by Selection

Soon followed domestication of the dog, horse, sheep, goat, ox and other animals thousands of years ago

Mating plants and animals with desirable traits resulted in selective breeding

39.

• Selective Breeding -selecting animals with desired characteristics to produce next generation(domestic animals: horses, cats, farm animals, crop plants)

40.

• • 1.Inbreeding -continued breeding of individuals with similar characteristics.Can create serious problems(bringing together 2 recessive alleles)

Albinism present in Hopi Indians.Only albino children are produced by albino parents.Two phenotypically normal individuals produce albino and non-albino children. 41.

• • 2.Hybridization -crossing dissimilar individuals to bring together the best of both organisms.(e.g. disease resistance and food-producing capacity)

Hereford, bred for meat production 42. Changes in whole sets of chromosomes

Polyploidyis the possession of more than2 completesets of chromosomes.

e.g.triploidmeans 3 sets;tetraploidmeans4 sets.

Formation of tetraploid offspring :

fertilization of diploid gametes or

whole set of chromosomes doubles after fertilization

43. 44. 45. Plant cloning: plant Tissue Culture A Requirement for Transgenic Development A plant partIs cultured Callus grows Shoots develop Shoots are rooted; plant grows to maturity 46.

• Cloning -a member of a population of genetically identical cells produced from a single cell

The adult sheep is Dolly, the first mammal cloned from an adult cell.The lamb is Dollys offspring, called Bonnie. 47. How Dolly was created 48. PROs

1 . Producing a greater understanding of the cause of miscarriages

2 . Generation of genetically modified animal organs

3. Preventing child suffering heredity

4. Cure damaged nervous system

49. CONs

1. Reducing genetic variability

2. Interfering with natural evolution

3.Risk of disease transfer

4 . Genetic tailoring of offspring

5. Detrimental effect on familial relationships

50. Genetics and Genetic Engineering 51. Genetic Engineering

technology involved in removing, modifying, or adding genes to a DNA molecule

52. Cloning DNA into a Plasmid Both plasmid and foreign DNA have stickyEco R I ends Insertion intoE. coli(transformation) Agar plates contain antibiotic. Grow at 37 C Place 1 colonyin liquid media + antibiotic.Grow at 37 C Purify Plasmid DNA (Billions of copies) 53. Restriction Endonucleases

Restriction endonucleases cut DNA

Present in bacteria

Cut at sequence specific sites

• Usually 4 or 6 base pairs long

Bacteria use them to destroy foreign DNA

Restriction enzymes can be purified and are used in genetic engineering studies

54. Restriction Endonucleases

Example Restriction enzymes

• Eco R I ( E .co li R estriction EndonucleaseI )

• StuI ( S treptomycestu bercidicusI)

GAATTC CTTAAG 5 5 3 3 Sticky EndedBlunt Ended AGGCCT TCCGGA 5 5 3 3 Eco R I StuI 55. Ligation of DNA Eco R I OH35PO 4 PO 4 53 OH T4 DNA Ligase StuI Circular DNA Eco R I G CTTAA AATTC G 56. Cloning DNA into Plasmids

Bacteria have a circular DNA genome

• 5 to 10 million base pairs (bp) in size

Many bacteria also contain plasmids

• Small circular DNA molecules, ~3,000 to 50,000 bp

• Note : The bacterial genome isnota plasmid

Plasmids contain extra genes which are often vital for the survival of the bacterium

• Nutrient metabolism, antibiotic resistance

Plasmids can be used as vectors in which foreign DNA can be ligated (cloned)

57. Transfer the vector to the host

• bacteria: electroporation

• Animal cells or plant cells: by infecting with a virus

• - bacteriophage

• - adenovirus

• - retrovirus

• (c) Gene therapy: using liposomes

58. Screening

Identification of successful transformed cells

E.g. using antibiotic resistant gene as the marker

59. Genetically modified (GM) food 60. Improving Plants and Animals

1988- first use of ice-minusfor Dutch Elm Disease

Long life tomatoes

BT Corn

Nitrogen fixing crops

61. Polymerase Chain Reaction (PCR)

Technique devised in 1983 to amplify small amounts of DNA

Can be performed on DNA from a single cell

- cigarette butt, a licked stamp, root of a single hair,1/50,000 a drop of blood (0.1 microliters)

The amplified DNA can then be used to:

- identify a suspect or victim

-determine an individuals sex

-determine species (if not human)

-parentage test

62. PCR to Amplify a Persons DNA

Steps Involved:

Isolate VNTRs from a persons DNA using restriction enzymes

Designprimers short segments of synthetic DNA that are complementary to DNAon either side of the VNTR regions

63.

Add vast excess of the primers and heat mixture to 75o C

This causes DNA strands to separate by breaking hydrogen bonds between bases

64. 4.Cool to 15o C.Primers hydrogen bond ( anneal ) to complementary strands 5.Add DNA polymerase and all four types of nucleotides.The polymerase (enzyme used in DNA replication) will fill in the rest of the two strands. 65. You now have two identical copies of the DNA you started with. 6. Repeat steps.Heat to break hydrogen bonds.Cool to anneal more primers (still there in vast excess).Allow DNA polymerase to fill in the remaining strands.Two strands of DNA become four.EtcEtcEtc.. 66.

Types of DNA probes:

RFLPs (restriction sites)

Repeated DNA

• Minisatellites(VNTRs = variable number tandem repeats)

• • Repeated units of 5 to several 10 bp

• Microsatellites(STRs = short tandem repeats)

• • Repeated units of 2-6 bp

• • 5-TAATAATAATAATAATAA-3

• • 3-ATTATTATTATTATTATT-5

67. How to make Antisense RNA mRNA and asRNA can form RNA/RNA duplex,which actually results in formation of 20-22 nts small RNA that trigger the degradation of mRNA transcribed region 5 control region 3 control region mRNA transcribed region 5 control region 3 control region Antisense mRNA (asRNA) 68.

DNA Sequencing

DNA sequencing= determining the nucleotide sequence of DNA.

Developed byFrederick Sangerin the 1970s.

69. DNA Fingerprinting

Used to identify individuals by their VNTR, RFLP, or STR regions

Steps involved:

Isolate and amplify DNA if needed

DNA is cleaved into smaller pieces with restriction enzymes

DNA is separated with gel electrophoresis

70.

DNA is transferred to a nylon membrane (blotting)

A radioactive primer is designed that will be complementary to unique regions (VNTR etc).Add this to nylon membrane containing DNA.

Wash off excess primer and hold nylon up to a photographic plate to expose.The pattern will be unique to the individual.

71.

How to fingerprint alleged paternity using microsatellites:

Extract DNA from mother, baby, and alleged father.

Synthesize oligonucleotide microsatellite primers and label one primer with fluorescent dye (2 primers per microsatellite).

Amplify microsatellites using PCR from mother, baby, father.

Electrophores microsatellite PCR products on a DNA sequencer (w/polyacrylamide) with a flourescent size standard loaded in the same lane or capillary.

3-4 different microsatellites can bemultiplexedin each lane or capillary by using 3-4 different fluorescent dyes.

Calculate size of each microsatellite relative to size standard (this size standard also can be run in the same gel lane or capillary using a 4th or 5th colored dye).

Sequence at least one copy of each allele to verify allele sizes.

72. SizeMother Baby Father

Paternity Analyses & Conclusions :

Baby and mother are expected to share on allele, and the baby and father the other allele.

If baby and father do not share a common allele, the father is not the father.

If the baby and father do share a common allele, paternity is possible, but not proven, because other men in the population also carry the allele at some frequency.

73. Forensics identify potential suspects whose DNA may match evidence left at crime scenes exonerate persons wrongly accused of crimes identify crime and catastrophe victims 74. Clearly, suspect one is the match.. If all RFLP and STR regions are considered, there is a one in 3.4 billion chance of error.This means theremaybe one other person on the planet that would be too similar to tell the difference. If other VNTR regions are also considered, the chances of error goway, waydown