Exam 2 Study Guide Flashcards
what is the purpose of DNA extraction
the removal of DNA from the cells or viruses in which it normally resides
what are the four steps that are common to all types of DNA extraction methods
- disruption
- lysis
- removal of proteins and contaminants
- recovery of DNA
Describe the basic technique/theory behind the preparation of crude lysates in DNA extraction
incubate at high temp or protein K
Be able to explain the basic technique/theory behind organic extraction methods and the disadvantages
use organic solvents to extract contaminants
disadvantage: time-consuming and uses toxic compounds
Be able to explain the basic technique/theory behind silica-based methods and the advantage(s)
uses adsorption of nucleic acids to a silica-gel membrane
advantages: only DNA is adsorbed, simpler and more effective
What is gel electrophoreses
Uses electrical current to separate different-sized molecules in a porous, sponge-like matrix
What is the purpose of electrophoreses
this process uses electricity to separate DNA and RNA fragments by size as they migrate through a gel matrix
What kind of gel is used in DNA electrophoreses
Agarose gel
what is blue tracking dye used for in gel electrophoreses? does it bind DNA?
Dye makes it easier to load the samples and visually
track the migration through the gel
Yes
explain how DNA moves through gel in gel electrophoreses
DNA and RNA molecules are negatively charged therefore electric current will move towards the positive pole
What is ethidium bromide
fluorescent dye to stain DNA/RNA
What is the purpose of the southern blot
used to detect specific DNA
molecules from among other molecules
Be able to name and describe the 5 steps of the southern blot
- Separating DNA fragments based on size via electrophoresis
- Transferring them to a membrane
- Hybridization with a labeled sequence-specific probe
- Washing
- Detection of labeled DNA band(s)
What kind of information does the southern blot give you
Reveals information about DNA: identity, size, and abundance
Describe how the southern blot probe is designed
single-stranded DNA
What has to be true about the sequence of the southern blot probe
it has to be complementary to the sequence you are testing
What is HRP and how does it provide a detection signal
horse radish peroxidase
catalyzes oxidation of luminol
How else can the southern blot probe provide a detectable signal
alkaline phosphatase or radioactive
What is the purpose/theory behind PCR
PCR enables the production, or amplification, of billions of copies of an original piece of DNA in a test tube within minutes or hours, not days
Who invented PCR
American biochemist Kary
Mullis,(1987)
What kind of information does PCR give you
information on a gene so you could make copies
What are primers and why are they necessary in PCR
A pair of primers with exposed 3′-OH groups that will bind to the particular sequence of interest in the DNA template
What are the necessary ingredients of a PCR reaction
DNA, taq polymerase, nucleotides, primer
Be able to name and describe the three main steps of PCR
- denaturation - breaking of double helix
- annealing - primers bind to complimentary strand
- extension - heat to create new DNA strands
What is a thermocycler
machine that cycles between the different necessary temperatures for PCR
What is Taq polymerase and what is the significance of this discovery
thermus aquaticus
since it’s heat-resistant you don’t need to add new polymerase during each cycle
What is the purpose/theory behind Real-time PCR (aka qPCR)
Allows you to copy a particular DNA sequence and quantify it simultaneously
What is the purpose of RNA isolation
allows you to see genes being transcribed
What is the main concern when isolating RNA molecules
RNA is unstable and has a short half-life
Which type of RNA represents gene expression
mRNA
What are Rnases
ribonuclease, type of nuclease that catalyzes the degradation of RNA
What are the basic steps in RNA isolation methods
- cell lysis
- inactivation of cellular nucleases
- separation of desired nucleic acid from cell debris
Explain the steps of RNA isolation using an organic solvent
- guanidinium thiocyanate
- extract homogenate
- RNA separated from DNA
- RNA is in aqueous phase
Explain the steps of RNA isolation using OligodT columns
- special column used with short chains
- mRNAs with poly(A) tails are added
- poly(A) tail pairs with chains
- rest of RNA passes through
- mRNA is washed using buffer that breaks bond
- leaving only mRNA with poly(A) tails
What is the purpose of Northern blotting
Hybridization technique for detection of specific RNA sequences
What are the basic steps of the Northern blot
- Total cellular RNA or mRNA is size-separated by denaturing agarose gel electrophoresis
- The separated RNA is transferred onto a nylon membrane
- The RNA is then detected with isotopic or non-isotopic labeled complementary DNA or RNA probe
What kind of information does the northern blot give you
- to observe a particular gene’s expression pattern between tissues
- detect overexpression of oncogenes and down-regulation of tumor-suppressor genes in cancerous cells
- determine the size of the product
What is the purpose of In Situ Hybridization
to identify the position of genes and localize and detect the specific DNA sequences in cells
What are the basic steps of In Situ Hybridization
- probe preparation and labeling
- tissue fixation
- permeabilization
- hybridization
- signal detection
What kind of information does In Situ Hybridization give you
precise localization of a specific segment of nucleic acid within a histologic section
What is the purpose of RT-PCR
determine how gene expression changes over time or under different conditions
What is reverse transcriptase
enzyme that makes DNA out of RNA
What is cDNA
complementary DNA
Describe the steps to make cDNA
- Separating mRNA molecules from other cellular RNA
- Using reverse transcriptase to copy the mRNA molecules into cDNA
What are antibodies
Important in protein isolation and
identification
How are antibodies made
derived from B-cells
What is an epitope
unique part of antigen recognized by antibody
What is a monoclonal vs. polyclonal antibodies
monoclonal = recognize one epitope
polyclonal = recognize multiple epitopes
How are monoclonal and polyclonal antibodies made
monoclonal = B-cell clone “hybridoma”
polyclonal = multiple B-cell clones
What are the advantages and disadvantages of monoclonal and polyclonal antibodies
poly may be cheaper but mono targets specific
What is the purpose of immunoprecipitation
Identification Of Protein-protein Interactions
What are the basic steps of immunoprecipitation
- Attach antibody to beads via protein A
- Lyse cells to release antigen and its binding partners
- Mix cell lysate + antibody-coated beads (antibody binds antigen)
- Purify antigen and its binding partners by centrifugation
What is the purpose of immunohistochemistry and immunofluorescence microscopy
visualize the location and distribution of proteins in cells
direct method
primary antibody only
indirect method
primary and secondary antibodies
enzyme-linked method
primary, secondary, and enzyme
What is the primary antibody specific to
antigen
What is the secondary antibody specific to
primary antibody
What is the purpose behind Western blotting
detect specific proteins from a complex mixture
What are the steps of western blotting
- electrotransfer
- antibody detection
- development
What kind of information does western blotting provide
protein expression and size
Describe the importance of the stacking gel? How does it work?
more refinement by doing a broad separation and then a more specific separation
How is the secondary antibody detected in western blotting
HRP enzyme
What is the purpose/theory behind ELISAs
detect antibodies
What does ELISA stand for
Enzyme Linked Immunosorbent Assay
What are the basic steps for ELISA
- add antigen/antibody to well
- add opposite
- add substrate
- measure light
direct ELISA
detects antigen
indirect ELISA
detects antibody
competitive ELISA
compares affinity
What kind of information does ELISA give you
depends on type of ELISA run
What is a standard curve
Serial dilutions of the cytokine being
measured
How do you make a standard curve and what information does it provide
diluting a sample down by a proportional amount to desired density
What are the advantages of ELISAs
easy due to wide variety of antibodies available and versatile
mouse female reproductive tract
two ovaries, fallopian tubes, leads to oviduct
meiosis
sexual cell division
oocyte development
begin divide inside fallopian tubes
fertilization occurs in the
fallopian tubes
what embryo stage in is the ovary
ovum
which embryo stages are in the fallopian tube
fertilization, zygote, morula
what embryo stage is in the uterus
blastocyst
blastocyst
multicelled zygote ready for implantation
inner cell mass
part of blastocyst, actual embryo
trophectoderm
cells of the outer layer are called this, these cells form the extraembryonic membranes, which are the placenta and allantois
pluripotent
have the potential to develop into every cell type in the fetus
basics of embryo production
6 - 12 oocytes per ovulation
estrous cycle = 4 - 5 days
Whitten effect
Exposing stock females to a sterile male mouse, or by introducing the soiled bedding of male mice into the cages of females to induce estrus
superovulation
injected with exogenous hormones prior to mating to induce larger litters
stud male
males used as mates for embryo to maximize sperm production
how to collect embryos
1 to 8 cell stages with syringe needle, dissect fallopian tube
embryo recipients
primed females exposed to sterile males
steps of designing and generating a transgene
- designing the DNA for insertion
- making the DNA in the lab
- injecting DNA into mouse embryos
concatemer
long continuous DNA molecule that contains multiple copies of the same DNA molecule linked together
GOI
gene of interest
promoter
a regulatory portion of the genome that helps to control transcription of a gene
reporter gene
way to check if the trans gene is expressed
plasmids
DNA vector, ex. drug resistance
first disease models
spontaneous mutations that happened to produce a medically relevant phenotype
What are the different ways to categorize a disease and why is that important to know when designing a disease model
- loss of function
- gain of function
- partial loss
importance - how to treat it
Be familiar with Nude mice and SCID mice and their importance as animal models for human disease
nude mice - good as tissue recipients
SCID - no immune system, good for studying “bubble boy” syndrome
forward vs reverse genetics
forward - identify the mutation and then work with it
reverse - make it freaky (mutate it)
steps of gene insertion using pronuclear injection
- microinject DNA into male pronucleus
- transfer to oviduct of pseudopregnant female
founders and their importance
the original DNA with transgene
important because you can go back and remake the population
does the presence of a transgene always mean that the disease phenotype you see is caused by the expression of that transgene
no
viruses used as vectors for genes
Moloney Murine Leukemia Virus - retrovirus
Lentivirus - disabled viral vector
transposons
segments of DNA that can be inserted into the genome by enzymes known as transposases
gene trap
transgene that stops other genes
International Gene Trap Consortium (IGTC)
institutions working towards creating mice for each individual gene
knock-out vs knock-in
knock-out = remove/inhibit gene
knock-in = give gene
homologous recombination and its role in KO/KI mice
A region of the cell’s genome has been replaced by a DNA fragment whose sequence is the same as, or very close to, the sequence of that region
Can cause the KO/KI
selectable cassette/selectable marker
gene for antibiotic resistance
how to create a transgenic mouse
- DNA construct
- insert into embryo
positive selectable markers
allows to select for cells that are positive for DNA
negative selectable markers
kills cells that express the DNA
how is electroporation used to generate targeted ES cells
allows DNA molecules to enter specific cells
chimeric mouse creation
an animal composed of cells from two different genetic lineages
is a chimeric mouse a transgenic mouse
no
How do you make/find your transgenic mouse after a chimera is made?
the chimera has to have litters
What are some of strategies used when mating your chimeric mice
use more embryonic stem cells, use phenotypic selectors
why are male chimeric mice used for breeding
males can impregnate multiple females
conditional knockouts
irreversible
can study germline
inducible knockout
reversible
can be isolated
Tet-On systems
tetracycline is injected or added to animal
Tet-Off systems
the transgene is active only in the absence of the antibiotic
constitutive vs conditional mutations
constitutive = present all the time
conditional = present sometimes, depends on another gene
Cre recombinase recognizes loxP sites
insert gene in between LoxP genes which pop out gene
Flp recombinase recognizes frt sites
place gene between Flp and frt, pops out gene
PhiC31 integrase recognizes attP and attB sites
brings in gene between attP and attB sites
What is the COIN system and how is it useful
” conditional by inversion”
can determine if gene causes a disease and if it can be reversed by restoring normal function
What are Floxed Stop Cassettes and how are they used
a short stretch of DNA that signals the end of gene transcription
and/or translation
shuts down expression of a gene, can be removable/reversible
What is gene editing and how does it differ from gene targeting
gene editing = more specific than gene targeting, more recent
Zinc-Finger Nucleases
zinc fingers recognize triplets to cause specific damage to cause DNA repair between the two fingers
TALENs
works similar to Zinc-Fingers, can only delete easier to design
CRISPR/Cas9
can add or remove DNA
advantages to gene editing
only needs a dozen fertilized eggs
any animal with eggs
only a few hundred base pairs of DNA
gene targeting benefits
cheaper than gene editing
