Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
55 Cards in this Set
- Front
- Back
|
What is the insert size for a fragment which uses a plasmid as a vector? What is a special feature? What is its host?
|
< 10 kb. Efficient cloning. Host is E. Coli.
|
|
|
What is the insert size for a fragment which uses a bacteriophage λ as a vector? What is a special feature?
|
10-20 kb. Size selection. Host is E. Coli.
|
|
|
What is a concatemer?
|
A long continuous DNA molecule that contains multiple copies of the same DNA sequence linked up.
|
|
|
What is a Cosmid vector?
|
Hybrids of λ phage and plasmids.
|
|
|
What is the insert size for a fragment which uses a Cosmid as a vector? What happens to most of the λ genome?
|
Up to 45kb. Most of the λ genome is deleted and replaced.
|
|
|
Is the recombinant phase still packaged and circularised?
|
Yes.
|
|
|
What does a Cosmid look like inside the host?
|
A large, circular plasmid.
|
|
|
What are two prerequisites cDNA inserts must have to be expressed in vitro?
|
Must be expressed using prokaryotic signals
Must be inserted in the correct orientation (or STOP codon may be reached too soon) |
|
|
Can expression (cDNA) libraries be screened with a specific antibody to find a gene?
|
Yes
|
|
|
A defect of which gene causes albinism?
|
Tyrosinase gene
|
|
|
What is the action of tyrosinase?
|
Converts tyrosine into a melanin precursor
|
|
|
How was the tyrosinase cDNA found? Step 1.
|
RNA converted to cDNA (from melanocyte cells)
|
|
|
How was the tyrosinase cDNA found? Step 2.
|
cDNA is cloned
|
|
|
How was the tyrosinase cDNA found? Step 3.
|
Hamster tyrosinase is purified.
|
|
|
How was the tyrosinase cDNA found? Step 4.
|
Antibody raised against the hamster tyrosinase.
|
|
|
How was the tyrosinase cDNA found? Step 5.
|
The antibody is made to bound to the protein produced by the specific clone which produces tyrosinase, and the clone is isolated.
|
|
|
What type of fragment is used in a T/A cloning vector? What is a special feature? What is its host?
|
PCR products are inserted. The special feature is that is direct. The host is E. Coli.
|
|
|
What type of fragment is used in an Expression vector? What is a special feature? What is its host?
|
cDNA is inserted. The special feature is that it produces protein. Host is E. Coli.
|
|
|
What is the insert size for a fragment which uses BAC as a vector? What is a special feature? What is its host? What does BAC stand for?
|
Up to 300 kb, special feature is that it is huge plasmid. Host is E. Coli. Bacterial artificial chromosome.
|
|
|
What is the insert size for a fragment which uses YAC as a vector? What is a special feature? What is its host? What does YAC stand for?
|
Up to 1 Mb, special feature is that it is a linear chromosome. Host is yeast. Yeast artificial chromosome.
|
|
|
What necessary property does partial digestion ensure?
|
Overlapping fragments
|
|
|
What are the 3 advantages of using YAC and BAC clones?
|
Reduce number of clones required for genomic library
Make ideal in situ hybridisation probes Can clone entire genes |
|
|
What is the first step to chromosome walking?
|
RFLP probes are hybridised to a genomic library.
|
|
|
What is the second step to chromosome walking?
|
Positive genomic fragments can be used as subsequent probes by subcloning distant ends (and so overlapping occurs)
|
|
|
What is are two features of chromosome walking?
|
Sequential hybridisation of large fragment clones.
Provides cloned fragments covering the candidate region. |
|
|
What type of blot can be used to find genes?
|
Zoo blots (blots running samples from different species)
|
|
|
Why is this a possibility?
|
Since genes are often conserved between species (but the fragment in question is highlighted at different points on the ladders between species)
|
|
|
What are the two main uses for cDNA?
|
Study and mutation detection
|
|
|
What are the range of techniques used to find the CFTR gene?
|
RFLP mapping, chromosome walking, Southern (zoo) and northern blotting, sequencing and screening of a cDNA library.
|
|
|
What is preimplantation genetic diagnosis?
|
Embryo selection based on genetic screening of IVF embryos.
|
|
|
What are the 3 steps of preimplantation genetic diagnosis?
|
Select embryos not carrying deleterious mutations
Select non aneuploid embryos Select sex |
|
|
What are tests are used for Down Syndrome, Severe Haemophilia and Huntingtons?
|
Down Syndrome: Karyotype
Haemophilia: RFLP Huntingtons: PCR |
|
|
What is FISH?
|
Fluorescene in situ hybridisation
|
|
|
What can karyotyping or FISHing be used to detect?
|
Chromosomes
|
|
|
What are the three methods of obtaining therapeutic proteins?
|
Protein isolation
Protein production 'Pharming' |
|
|
What is Pharming?
|
The process of genetically modifying plants or animals to they produce substances which can be used in pharmacy.
|
|
|
What does 'transgenic'?
|
An organism with foreign DNA stably integrated into its genome.
|
|
|
How can Factor IX be expressed in transgenic sheep?
|
Removing lactoglobulin gene (not the promoter), inserting the human Factor IX gene. Exposing it to high lactose levels, causing more DNA Pol to bind to the promoter, and produce Factor IX. Put this in sheep oocyte, growth and development, milk transgenic sheep and purify proteins.
|
|
|
What is gene therapy?
|
Genetic modification of the patient's DNA.
|
|
|
How are transgenic humans made?
|
Germ Line gene therapy.
|
|
|
What are the 3 types of targeted gene modification systems?
|
Zinc Finger Nucleases (ZNF)
Transcription activator-like effector nucleases (TALEN) Clustered regularly interspaced short palindromic repeats (CRISPR) |
|
|
What does gene editing aim to do?
|
Specifically alter genes via targeted double strand breaks.
|
|
|
What do all the targeted gene modification systems provide?
|
Programmable nuclease activity directed to a specific DNA sequence via DNA-binding proteins or guide RNAs.
|
|
|
What is one method of targeted gene inactivation involving RNA?
|
RNA interference (RNAi)
|
|
|
What is RNA interference?
|
A highly conserved mechanism whereby double stranded RNA induces destruction of the complementary mRNA.
|
|
|
How is RNAi used experimentally used?
|
Silence gene expression (short dsRNA). Can be used to switch off certain alleles.
|
|
|
What type of potency are embryonic stem cells? Where are they derived from?
|
Pluripotent cells. Derived from the blastocyst.
|
|
|
Are pluripotent stem cells capable of self renewal? In what cell type can they differentiate into?
|
They are capable of self renewal and they can differentiate into all cell type.
|
|
|
What are the 3 main problems associated with pluripotent stem cells being implanted in people?
|
Immunogenicity (rejected tissue)
Embryo destruction (ethics) Cancerous structures |
|
|
Are patient specific pluripotent stem cells possible? If so, how are they bought about?
|
They are possible, undergo targeted gene alterations or stem cell therapy. Cloning is carried out by nuclear transfer.
|
|
|
What are the 6 steps of cloning by somatic cell nuclear transfer?
|
1: Take unfertilised egg
2: Remove the nucleus 3: Take adult mammary cell 4: Donate the mammary nucleus to the egg 5: Implant the embryo into a surrogate 6: Clone born! |
|
|
What is the difference between reproductive and therapeutic cloning?
|
Reproductive cloning forms an identical individual
Therapeutic cloning forms stem cells for therapy |
|
|
How can mitochondrial disease be avoided?
|
Through pronuclear transfer.
|
|
|
What is an induced pluripotent stem cell?
|
A normal cell that has been reprogrammed to be pluripotent.
|
|
|
What kind of solution can this be done with?
|
Reprogramming transcription factor cocktail.
|
