Restriction Mapping and Knockout Flashcards
(25 cards)
Prior to sequencing a large piece of DNA what is typically done to locate markers in the DNA?
Preliminary mapping
When restriction enzymes are mapped what results?
A restriction Map
What is the uses for restriction mapping?
To determine if a piece of insert DNA has gone into a vector in a particular orientation
What does site directed mutagenesis accomplish?
It is a molecular tool that provides a means to alter a single or several SPECIFIC bases in a gene to a base of your choosing.
In site directed mutagenesis how and by what process is the codon changed?
The codon for a specific amino acid is mutagenized or changed to a codon for some other amino acid. PCR is a convenient way to introduce these changes.
Give an example of an experiment where site directed mutagenesis is helpful.
You want to alter the base sequence in a cloned gene from TAC codon for tyrosine is changed to TTC codon for phenylalanine. WHY? The primary sequence of proteins effects the 3D structure of proteins thus altering function. Tyr has the same structure as Phe except Phe is missing the hydroxyl group on phenolic ring. This experiment will determine if the presence of the hydroxyl group (structure) is critical to the protein function.
What does PCR site-directed mutagenesis take advantage of?
- genes cloned in E coli are methylated on
5’-GATC-3’ sequences, and DNA made from PCR reactions is not methylated. - The methylated 5’-GATC-3’ sequence is recognized by the restriction enzyme Dpnl.
- EXTREMELY high fidelity DNA can and must be employed in the PCR reaction.
IN the site mutagenesis reaction example what high fidelity DNA polymerase used? And why is it used?
Pfu Polymerase- Pyrococcus furiosus
It is used bc it is very heat stable and very faithful and has proofreading activity.
You ___ __ want to introduce a single _____ change in the DNA you are mutagenizing
do not
unwanted
WHat is a gel motility shift?
An assay for DNA-protein interactions
In what cases will protein interact directly with DNA?
- RNA polymerase must recognize and bind to ver specific sequences of DNA known as promotors
- Various transcription factors must recognize and bind to specific DNA sequences
When the protein binds to a small piece of DNA how is the motility changed? And what is caused because of this on the gel electrophoresis?
The DNA + Protein has a much lower mobility in gel electrophoresis than the ‘naked’ DNA
A band shift is caused.
How does a “supershift” occur?
When the same DNA binds to 2 molecules of protein that effect the band shift seen on the gel motility
In a supershift what is the second protein made of?
It can be different form the first same as the first or an antibody.
WHy do a gel motility shift?
To see if the protein binds to the DNA
WHy are knockouts done?
To disrupt or alter a particular gene in a living organism
WHat do knockouts allow?
They allow for the investigation of a missing gene or otherwise altered gene in the overall functioning of the organism
What organism is mostly used in knockout experiments and why?
MIce. The product mice are called knockout mice. They are a good choice because of such a short generation time
How do you perform a knockout experiment?
- Begin with cloned DNA with the gene to be knocked out
- The gene is interrupted with a gene for neomycin resistance.
- In a location other than within the gene, a THYMIDINE KINASE gene is inserted.
- The altered mouse gene is mixed with brown mice embryonic cells
- In some of the cells the interrupted gene will enter the nucleus and HOMOLOGOUS RECOMBINATION between the intact gene and altered gene will occur
- The recombination event inserts the altered gene into the mouse genome removes the thymidine kinase gene
- Eliminate the cells in which homologous recombination did not occur by growing the cells in a medium containing a neomycin derivative
- Eliminate cells in which non specific recombination occurred by placing them in the presence of gangcyclovir
- Following treatment with the neomycin analog and gangcyclovir, the stem cells that remain have undergone homologous recombination and are heterozygous for the interrupted target gene
- Stem cells are injected into a black mouse blastocyst
- the embryo is placed into a surrogate mother mouse
- The baby mouse is a chimera!
WHat are the extra genes added into the cloned DNA during a knockout experiment and what do they allow?
thymidine kinase and neomycin resistance
they allow for the identification and removal of clones in which the targeted gene was not disrupted
What are the three possibilities that can occur after the altered mice DNA is mixed with the brown mice stem cells?
- homologous recombination will occur between the intact gene and target gene and thymidine kinase gene is removed
- recombination will not occur at all and no interruption of the resident gene occurs
- nonspecific recombination occurs and the altered gene is inserted randomly into the mouse genome without interrupting the target gene
What does the drug gangcyclovir do?
It is lethal to tk+ cells (thymidine kinase positive)
remember that the thymidine kinase gene is removed when homologous recombination occurs but not removed when recombination is non-specific
What is a chimera?
An individual consisting of tissues of a diverse genetic constitution
WHat happens after the chimera is created?
The chimera is allowed to mature assuming the knockout is not lethal. It is mated with a black mouse. Brown (from chimera) is dominant so some of the offspring will be brown. The offspring carrying the interrupted gene are mated and the offspring should be homozygous for the interrupted gene. Now the phenotypic difference by the knockout gene can be discovered!
