a. Genome projects: improve knowledge/understanding/diagnosis/treatment of genetic disorders
Human Genome Project 1990
- Identify genes in human genome & their loci
- Determine sequence of 3billion base pairs in human DNA & store in database
- Address arising ethical/legal/social issues
Number of genes in human genome – 20,500
Large number of repeating sequences
Sanger Sequencing – very slow
- DNA polymerase, DNA, nucleotides
- ddNTP + fluorescent marker: altered nucleotide (lacks OH = chain-terminating)
= different length strand each time base reached
- Gel electrophoresis + x-ray film: separates strands + expose banding pattern = sequence
100K Genome Project 2012
- Sequence 100,000 genomes & study variation
- Enable medical & scientific discovery
o Locate genes responsible for rare genetic disorders by comparing sufferer’s with normal genome –
predisposition to diseases
- Develop UK genomics industry
Next Generation Sequencing – faster/cheaper/reliable/accurate: sequences entire genome in few hours
b. Ethical issues of testing – can outweigh benefits
- ownership of genetic information / misuse of data
o passed to insurance companies / police
o not safe: computer storage system can be hacked
- risk of discrimination / social stigmatisation
o ancestral relationships
Routine screening for adult onset disorders: Alzheimer’s / cancer
- when to tell screened children they have predisposition for adult onset disorder?
Screening embryos can detect genetic disorders: cystic fibrosis / Huntington's disease / thalassaemia
- could lead to abortion / people choosing alleles for specific characteristics – designer babies
Genetic screening: scientists scan patient’s DNA for mutated sequences – compare sequence in gene to normal version
Genetic counselling: provide info/advice to people at risk of genetic disease – help make informed decisions
c. sequencing other organisms’ genomes
- investigate evolutionary relationships for classification
- conserve species
- study disease-causing organisms – help find treatments/preventions
Anopheles gambiae mosquito + plasmodium parasite (principal vector of malaria)
Mosquito: evolved insecticide resistance – hampers attempts to eradicate malaria
allows scientists to develop chemicals – restore insecticide susceptibility
Plasmodium: developed multi-drug resistance
allows development of more effective drugs
, d. genetic fingerprinting = amplify quantity of DNA for analysis & produce DNA profile
- exons – code for proteins
- introns – non-coding: Short Tandem Repeats / microsatellites / Hyper Variable Region
o number of STRs: inherited – individuality = variation
- human chromosome 7: D7S280 STR = ‘GATA’ bases repeat 6-15 times
1. Polymerase Chain Reaction: amplify DNA – synthesise complementary strands to short fragments (800 bases)
1. denaturation – heat 95˚C: break H-bonds & separate strands
2. annealing – cool 50-60˚C: allow primer to bind to DNA
primer: short DNA fragment complementary to start of DNA sequence
3. extension – heat 70˚C: allow Taq polymerase to add complementary nucleotides to primer
Taq polymerase: heat-stable DNA polymerase from hot spring bacterium
repeat: 40 cycles = 1billion+ copies
Limitations
- contamination amplified
- taq polymerase errors accumulate – can’t proofread+correct mistakes
- can only amplify small fragments
- enzymes sensitive to inhibitors
- efficiency decreases: reagent concentration < product
2. Restriction endonuclease enzyme: cut out STRs = cut DNA into fragments
3. Gel Electrophoresis: isolate & separate DNA fragments based on size (number of repeats)
- agarose gel – contains pores in matrix
- samples loaded into wells at negative electrode – voltage applied across gel
o negative charge in DNA phosphate group = attracted to positive electrode
o smaller fragments migrate further – easier through gel pores
- estimate fragment size: compare to DNA ladder (contains fragments of known size)
4. Make DNA visible
- Southern blotting: transfer DNA to nylon membrane
- add radioactive/florescent probes: detect specific sequence – bind to complementary STR (DNA hybridisation)
- place on X-ray film & expose to X-ray – autoradiograph reveals banning pattern
use of genetic fingerprinting – non-invasive (saliva/urine/hair – not blood) + can use small samples
- forensic evidence to identify/eliminate suspects / identify human remains
o could be present but not criminal
- prove paternity / siblings
- identify relatives for immigration purposes
- phylogenetic studies: species relatedness
Human Genome Project 1990
- Identify genes in human genome & their loci
- Determine sequence of 3billion base pairs in human DNA & store in database
- Address arising ethical/legal/social issues
Number of genes in human genome – 20,500
Large number of repeating sequences
Sanger Sequencing – very slow
- DNA polymerase, DNA, nucleotides
- ddNTP + fluorescent marker: altered nucleotide (lacks OH = chain-terminating)
= different length strand each time base reached
- Gel electrophoresis + x-ray film: separates strands + expose banding pattern = sequence
100K Genome Project 2012
- Sequence 100,000 genomes & study variation
- Enable medical & scientific discovery
o Locate genes responsible for rare genetic disorders by comparing sufferer’s with normal genome –
predisposition to diseases
- Develop UK genomics industry
Next Generation Sequencing – faster/cheaper/reliable/accurate: sequences entire genome in few hours
b. Ethical issues of testing – can outweigh benefits
- ownership of genetic information / misuse of data
o passed to insurance companies / police
o not safe: computer storage system can be hacked
- risk of discrimination / social stigmatisation
o ancestral relationships
Routine screening for adult onset disorders: Alzheimer’s / cancer
- when to tell screened children they have predisposition for adult onset disorder?
Screening embryos can detect genetic disorders: cystic fibrosis / Huntington's disease / thalassaemia
- could lead to abortion / people choosing alleles for specific characteristics – designer babies
Genetic screening: scientists scan patient’s DNA for mutated sequences – compare sequence in gene to normal version
Genetic counselling: provide info/advice to people at risk of genetic disease – help make informed decisions
c. sequencing other organisms’ genomes
- investigate evolutionary relationships for classification
- conserve species
- study disease-causing organisms – help find treatments/preventions
Anopheles gambiae mosquito + plasmodium parasite (principal vector of malaria)
Mosquito: evolved insecticide resistance – hampers attempts to eradicate malaria
allows scientists to develop chemicals – restore insecticide susceptibility
Plasmodium: developed multi-drug resistance
allows development of more effective drugs
, d. genetic fingerprinting = amplify quantity of DNA for analysis & produce DNA profile
- exons – code for proteins
- introns – non-coding: Short Tandem Repeats / microsatellites / Hyper Variable Region
o number of STRs: inherited – individuality = variation
- human chromosome 7: D7S280 STR = ‘GATA’ bases repeat 6-15 times
1. Polymerase Chain Reaction: amplify DNA – synthesise complementary strands to short fragments (800 bases)
1. denaturation – heat 95˚C: break H-bonds & separate strands
2. annealing – cool 50-60˚C: allow primer to bind to DNA
primer: short DNA fragment complementary to start of DNA sequence
3. extension – heat 70˚C: allow Taq polymerase to add complementary nucleotides to primer
Taq polymerase: heat-stable DNA polymerase from hot spring bacterium
repeat: 40 cycles = 1billion+ copies
Limitations
- contamination amplified
- taq polymerase errors accumulate – can’t proofread+correct mistakes
- can only amplify small fragments
- enzymes sensitive to inhibitors
- efficiency decreases: reagent concentration < product
2. Restriction endonuclease enzyme: cut out STRs = cut DNA into fragments
3. Gel Electrophoresis: isolate & separate DNA fragments based on size (number of repeats)
- agarose gel – contains pores in matrix
- samples loaded into wells at negative electrode – voltage applied across gel
o negative charge in DNA phosphate group = attracted to positive electrode
o smaller fragments migrate further – easier through gel pores
- estimate fragment size: compare to DNA ladder (contains fragments of known size)
4. Make DNA visible
- Southern blotting: transfer DNA to nylon membrane
- add radioactive/florescent probes: detect specific sequence – bind to complementary STR (DNA hybridisation)
- place on X-ray film & expose to X-ray – autoradiograph reveals banning pattern
use of genetic fingerprinting – non-invasive (saliva/urine/hair – not blood) + can use small samples
- forensic evidence to identify/eliminate suspects / identify human remains
o could be present but not criminal
- prove paternity / siblings
- identify relatives for immigration purposes
- phylogenetic studies: species relatedness
