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Molecular Genetics > Gene Manipulation > Flashcards

Gene Manipulation Flashcards

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1
Q

Why is the human genome sequenced?

A
  • Fear it would end
  • Labour intense sequencing techniques would sacrifice scientists’ time
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2
Q

The inheritance of _______ is transmitted by _____ and _____ are made of ______

A
  • Characters
  • Genes 2 times
  • DNA
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3
Q

Why are single genes isolated?

A
  • To learn from its sequence
  • To modify genes and study its function
  • To move genes between organisms by transgenics
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4
Q

How can a gene be isolated from DNA?

A
  • By using a cell system, cloning DNA
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5
Q

Explain the process of DNA cloning

A
  • 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
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6
Q

What are the two types of DNA sources?

A
  • Genomic DNA (contains ALL DNA, exons, introns etc)
  • cDNA (is a copy DNA from mature RNA)
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7
Q

what enzyme makes cDNA?

A

reverse transcriptase

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8
Q

What are restriction enzymes?

A
  • Enzymes that recognize palindromic DNA sequences and chop them up
  • The fragments are cut with sticky ends to ligate unto vectors
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9
Q

What makes a vector?

A
  • An origin of replication (ori)
  • A selectable marker to identify recombinant (ampR)
  • At least one RE cleavage site to insert donor DNA (EcoRI)
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10
Q

how are vectors ligated with donor DNA?

A

By sequence complementation

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11
Q

Explain the process of creating recombinant in detail

A
  • 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
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12
Q

List some other types of vectors

A
  • Viruses (can carry a 10Kb donor)
  • Plasmids
  • Cosmids (can carry a 45Kb donor, replicate as plasmids)
  • Artificial chromosomes (can clone full genomes without chopping)
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13
Q

What developments took place in the 1970s that allowed the geneticists to manipulate DNA?

A
  • Cloning
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14
Q

what were some of the problems and solutions during the early stages of the human genome project?

A

Problems: sequencing was labour intense, slow, and costly
solutions: Milestones were set up and sequencing technologies were developed

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15
Q

List some examples of different markers for selection and cloning

A
  • ampicillin
  • LacZ
  • Antibiotics
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16
Q

Explain the main steps of PCR

A
  • 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)
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17
Q

what are probes?

A

Probes are pieces of labelled DNA used to find a complementary DNA fragment in a mixture of total cellular DNA

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18
Q

How can probes be used to screen a library?

A
  • 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
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19
Q

explain how to isolate a disease allele with cloning

A
  • 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
20
Q

Explain sanger sequencing

A
  • 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)
21
Q

what is the function of ddNTPs?

A

they cannot form phosphodiester bonds with the next nucleotides so they terminate the sequence extension

22
Q

what are the difference between sanger and automated sequencing

A
  • 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
23
Q

What is shotgun sequencing?

A
  • 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
24
Q

What are the similarities between the three main massive parallel sequencing technologies?

A
  • 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)
25
Q

What are unique features of pyrosequencing

A
  • 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
26
Q

What is a unique feature of illumina

A
  • DNA molecules are amplified on a slide, labelled nucleotides are kept and non-labelled ones are washed away (bridge amplification)
27
Q

what are unique features of SOLID

A
  • 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.
28
Q

What are characteristics of PacBio and Nanopore

A

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

29
Q

What has been the impact of massive parallel sequencing during COVID pandemic?

A

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

30
Q

List some examples of of transgenic plants

A
  • Rice that produces vitamin A
  • Soybeans that produce mono saturated fatty acids
  • Trangenic plants producing vaccine products
31
Q

What are some challenges in the transgenics with the rice plant in ß-carotene pathway

A
  • 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
32
Q

How is transgenics used to study gene expression?

A
  • 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
33
Q

How can u detective the presence of a transgene

A
  • Extracting DNA from progeny
  • run a PCR for gene of interest
  • Run in a gel
34
Q

How are knockouts done in mice?

A
  • 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
35
Q

what is the advantage of conditional knockouts?

A

Conditional knockout mice survive longer

36
Q

Explain RNAi

A
  • 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
37
Q

Explain CRISPR

A
  • ## Cluster of Regularly Interspaced Short Palindromic Repeats
38
Q

What are uses of CRISPR?

A
  • Knockout genes in live organisms
  • Blocking and activating gene expression with cas9
  • Precise gene knockout to study gene function
  • Inducing epigenetic change
39
Q

What was done in germline CRISPR editing in cattle?

A

A CRISPR edited calf was born with the SRY gene on makes edited into its chromosome 17 making COSMO physiologically a male

40
Q

What are the two types of gene therapy?

A

Somatic gene therapy
Germline gene therapy

41
Q

What is somatic gene therapy

A

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

42
Q

What is germ-line gene therapy

A
  • Correcting the disease in question and transmitting the normal genotype TO the progeny
  • mitochondrial diseases
43
Q

What are viral vectors used in gene therapy?

A
  • Retrovirus: Attacks proliferating cells and can integrate into patient genome
  • Adenovirus: attacks non dividing cells and doesn’t integrate into patient genome
44
Q

What are the four factors that makes ADA-SCID a good candidate for gene therapy

A
  • 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
45
Q

Explain mitochondrial germ line therapy

A
  • 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
46
Q

What is therapeutic cloning?

A

The creation of the early stage embryos to harvest stem cells for the treatment of disease

47
Q

What is reproductive cloning?

A
  • The creation of an animal
  • The most serious concerns are developmental defects

Molecular Genetics (2 decks)

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