Gene Manipulation Flashcards
Why is the human genome sequenced?
- Fear it would end
- Labour intense sequencing techniques would sacrifice scientists’ time
The inheritance of _______ is transmitted by _____ and _____ are made of ______
- Characters
- Genes 2 times
- DNA
Why are single genes isolated?
- To learn from its sequence
- To modify genes and study its function
- To move genes between organisms by transgenics
How can a gene be isolated from DNA?
- By using a cell system, cloning DNA
Explain the process of DNA cloning
- Extract the DNA from the organism under study
- Cut the genome DNA into pieces by restriction enzymes
- Isolate a vector and open it with the same RE
- Insert DNA into vector = recombinant
- Insert into a cell system to produce clones
- Identify clone carrying DNA of interest
What are the two types of DNA sources?
- Genomic DNA (contains ALL DNA, exons, introns etc)
- cDNA (is a copy DNA from mature RNA)
what enzyme makes cDNA?
reverse transcriptase
What are restriction enzymes?
- Enzymes that recognize palindromic DNA sequences and chop them up
- The fragments are cut with sticky ends to ligate unto vectors
What makes a vector?
- An origin of replication (ori)
- A selectable marker to identify recombinant (ampR)
- At least one RE cleavage site to insert donor DNA (EcoRI)
how are vectors ligated with donor DNA?
By sequence complementation
Explain the process of creating recombinant in detail
- Use the same RE to chop donor DNA and open up vector with sticky ends
- Ligate donor DNA to vector with DNA ligase
- Recombinant plasmids will not be resistant to what they were resistant to before
- If placed on an agar with certain marker, recombinant bacteria will be killed and non recombinant will grow
List some other types of vectors
- Viruses (can carry a 10Kb donor)
- Plasmids
- Cosmids (can carry a 45Kb donor, replicate as plasmids)
- Artificial chromosomes (can clone full genomes without chopping)
What developments took place in the 1970s that allowed the geneticists to manipulate DNA?
- Cloning
what were some of the problems and solutions during the early stages of the human genome project?
Problems: sequencing was labour intense, slow, and costly
solutions: Milestones were set up and sequencing technologies were developed
List some examples of different markers for selection and cloning
- ampicillin
- LacZ
- Antibiotics
Explain the main steps of PCR
- template DNA strands are separated at a high temperature (denaturation)
- ssPrimers are added to one template strand (annealing)
- DNA TAQ polymerase incorporates free nucleotides (extension)
what are probes?
Probes are pieces of labelled DNA used to find a complementary DNA fragment in a mixture of total cellular DNA
How can probes be used to screen a library?
- A probe is made from the gene of interest that’s closest to a marker
- DNA is inserted into vector and put on a gel
- Then, a new probe is used to do the whole process again
explain how to isolate a disease allele with cloning
- Know the position of the gene of interest (disease allele) on genetic map
- If allele is very often inherited with a random molecular marker then the RMR is close to the allele
- probes from markers with disease gene are used to screen libraries
- human DNA fragments within mapped region is extracted
- note if DNA fragments have changes over time, if they have what tissue was affected by this change
Explain sanger sequencing
- Four different reactions with ddNTPs are run
- each reaction is terminated at different lengths depending of the ddNTP in that reaction
- Reactions are run in a gel and read from small fragments to large fragments (bottom to top)
what is the function of ddNTPs?
they cannot form phosphodiester bonds with the next nucleotides so they terminate the sequence extension
what are the difference between sanger and automated sequencing
- Instead of running each 4 reactions in different tubes, all four are placed in one tube
- A different fluorescent dye is used to label the ddNTPs in gel
- the automated DNA sequencing machines run more reactions at a faster pace
What is shotgun sequencing?
- the whole genome is chopped up and cloned
- ssDNA is sequenced from templates
- overlapping regions on different short sequences are identified
- Sequence accuracy is increased by repeated sequencing to confirm
What are the similarities between the three main massive parallel sequencing technologies?
- All libraries are made by DNA fragmentation and lighting to sequence adapters
- Amplification is done by emulsion PCR
- All sequences are run at the same time
- They have a shorter read length (200Kb) than automated sanger sequencing (600Kb)
What are unique features of pyrosequencing
- It can’t incorporate nucleotides without pyrophosphate (PPi)
- PPi produces light with luciferase, the amount of light produced is proportional to the amount of nucleotides produced
What is a unique feature of illumina
- DNA molecules are amplified on a slide, labelled nucleotides are kept and non-labelled ones are washed away (bridge amplification)
what are unique features of SOLID
- ligation sequencing by DNA ligase
- The sequence of the DNA is determined by ligation of fluorescently labeled DNA probes using the DNA on the beads as a template.
What are characteristics of PacBio and Nanopore
PacBio: they have long sequencing reads to increase accuracy. Adaptors on the end of each DNA fragment
Nano-pore: Sequence of DNA is passed through a small pore and each nucleotide have a different disruption to be read on the machine
What has been the impact of massive parallel sequencing during COVID pandemic?
we were was able to sequence a virus as it circulates, to show that the variant of COVD was more prevalent and it revealed mutations related to increased transmission
List some examples of of transgenic plants
- Rice that produces vitamin A
- Soybeans that produce mono saturated fatty acids
- Trangenic plants producing vaccine products
What are some challenges in the transgenics with the rice plant in ß-carotene pathway
- The rice strain used did not grow as well as local rice varieties
- Rice strains with the β-carotene-producing genes had
foreign genes insertions in genome locations that caused failure in thriving - ß-carotene is only beneficial for human consumers and not for the plant itself
How is transgenics used to study gene expression?
- by fusing a reporter gene (lacZ) with a promoter on genome map to detect the presence of a trans gene
- by inserting vector with gene of interest into embryo and observe progeny phenotype
How can u detective the presence of a transgene
- Extracting DNA from progeny
- run a PCR for gene of interest
- Run in a gel
How are knockouts done in mice?
- Modify form of gene linked to genes by crossing over
- Recombinants and non-recombinants are grown on a culture
- a positive (selected for) and counter selection (selected against) is performed on genes on interests
- A clonal line with disrupted or knockout version of gene of interest is created
what is the advantage of conditional knockouts?
Conditional knockout mice survive longer
Explain RNAi
- a dsRNA is delivered into a cell and cleaved by Dicer
- the dsRNA fragments (RNAi) can inhibit gene expression with complementary sequence of RISC
- A specific gene can be targeted and shut down by delivering dsRNA
Explain CRISPR
- ## Cluster of Regularly Interspaced Short Palindromic Repeats
What are uses of CRISPR?
- Knockout genes in live organisms
- Blocking and activating gene expression with cas9
- Precise gene knockout to study gene function
- Inducing epigenetic change
What was done in germline CRISPR editing in cattle?
A CRISPR edited calf was born with the SRY gene on makes edited into its chromosome 17 making COSMO physiologically a male
What are the two types of gene therapy?
Somatic gene therapy
Germline gene therapy
What is somatic gene therapy
Cells are removed from a patient and they’re made transgenic by introducing the wild type gene
- The defect wont be passed to progeny
- Immuno deficiencies and blindness
What is germ-line gene therapy
- Correcting the disease in question and transmitting the normal genotype TO the progeny
- mitochondrial diseases
What are viral vectors used in gene therapy?
- Retrovirus: Attacks proliferating cells and can integrate into patient genome
- Adenovirus: attacks non dividing cells and doesn’t integrate into patient genome
What are the four factors that makes ADA-SCID a good candidate for gene therapy
- The gene was cloned and well characterized
- the blood cells can be gotten from patients and reintroduced with fxnal copies of gene easily
- Small amounts of the fxnal ADA gene restores partial immune fxn
- ADA overproduction has no toxic effects
Explain mitochondrial germ line therapy
- This is a process that transfers the spindle chromosomal complex between the donor and recipient cells
- Isolates and transplants the chr. from a patients oocyte to the cytoplasm of another egg with mtDNA
- Viable approach to avoid mitochondrial diseases
What is therapeutic cloning?
The creation of the early stage embryos to harvest stem cells for the treatment of disease
What is reproductive cloning?
- The creation of an animal
- The most serious concerns are developmental defects
