- 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
- Genetic engineering and GMO
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 affects 1 in every 3,000 births
- Cause: an inherited autosomal recessive gene
- 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
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.
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- Ifher wife doesn't have family history of CF disease, the
chance of their offspring having the disease is rather low.
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- Ifher wife has family history of CFdisease, she need to know
whethershe is a carrier.
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- 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
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
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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.
- Mostly males (most common form is X-linked)
- Cause: ADA (adenine deaminase) deficiency
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
- 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
- June 2000 completion of a working draft of the entire human
genome
- February 2001 analyses of the working draft are published
inNature
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.
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- Selective Breeding -selecting animals with desired
characteristics to produce next generation(domestic animals:
horses, cats, farm animals, crop plants)
40.
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- 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.
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- 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.
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- 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
- Cut at sequence specific sites
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- 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
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- Eco R I ( E .co li R estriction EndonucleaseI )
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- 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
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- 5 to 10 million base pairs (bp) in size
- Many bacteria also contain plasmids
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- Small circular DNA molecules, ~3,000 to 50,000 bp
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- Note : The bacterial genome isnota plasmid
- Plasmids contain extra genes which are often vital for the
survival of the bacterium
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- 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
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- bacteria: electroporation
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- Animal cells or plant cells: by infecting with a virus
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- (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
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)
62. PCR to Amplify a Persons DNA
- 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.
- RFLPs (restriction sites)
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- Minisatellites(VNTRs = variable number tandem repeats)
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- Repeated units of 5 to several 10 bp
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- Microsatellites(STRs = short tandem repeats)
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= 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
- 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