Theisen and Costa Review Flashcards
Primary cell cultures divide only a limited number of times before losing their ability to proliferate. What is this called?
senescence
note: these cell lines are known as finite
Where are primary cells derived from? How are they cultured?
animal tissue; cultured either as tissue explants or single cells
Do primary cells start as a heterogenous or homogenous population?
heterogenous
These type of cells are characterized by spontaneous genetic mutation or by transformation vectors and have an infinite life span, are easy to grow and preserve
immortal cells
ex: HeLA cells
Artificial expression of this key protein that prevents degradation of chromosome ends during replication is found in immortal cells
telomerase
What is the cell type and where do the following cell lines come from:
L6 PC12 SP2 R1 H1, H9
L6: myoblast - rat PC12: chromaffin cell - rat SP2: plasma cell R1: embyronic stem cell-human H1, H9: embryonic stem cell - human
SDS is largely ___ with a single ___ charge
hydrophobic; negative
What does SDS do?
unfolds proteins and allows for all proteins to migrate towards positive charge in an SDS - PAGE gel
What are the steps of western blotting?
- resolve protein samples on native PAGE
- elcetrophoreticlally transfer fractionated proteins from gel onto PVDF membrane
- Block the membrane with neutral protein
- incubate the membrane with HRP-labeled antibody specific to prey protein
- incubate the blot with chemiluminescent HRP substrate and expose to film
This detection method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable.
indirect immuno-cytochemsitry
What is mass spec used for?
identify unknown proteins and post-translational modifications
What does mass spec require?
tryptic digestion products (peptide fragments), ionization (charge) a detection method (time of flight) and computer databases with known protein sizes
What is the difference between standard mass spec and tandem mass spec?
in standard mass spec the ion source goes straight into the mass analyzer before detection
in tandem mass spec there is a mass filter and then fragmentation and then the mass analyzer before detection
In regards to analyzing interacting proteins, immunoprecipitation can do two things; what are they?
can isolate one protein from a mixture of many proteins
can find “co_IP” partners to identify protein complexes
Proteins can be detected and quantitated by ELISA, the presence of the colored product indicates what?
presence of the antigen
Which ELISA is used for the HIV test?
Indirect ELISA
In regards to analyzing DNA, what is the difference between an agarose gel from an SDS-PAGE?
the DNA is already charged
Why is a plasmid circular?
because bacterial genome is circular so it will recognize itself
What are cloning vectors used for?
transforming bacteria; carry and replicate manipulated gene products
What are cDNA clones?
DNA copy of mRNA: no introns, much smaller than original gene, and requires reverse transcriptase
What is the difference between genomic library and cDNA library?
genes vs gene products
What is FISH and what is it used for?
RNA probes; can be used to analyze the presence and location of genes (cytogenetics)
What is the series of events that take place in pcr?
heat strands to denature,
cool to anneal primers, DNA synthesis; cycles over and over again
What is CSI and what is it used for?
similar to pcr, utilizes naturally occurring short tandem repeats (STR); used in forensics
When is real time pcr used?
gene expression analysis, (cancer research and drug research) disease diagnosis and management, food testing, animal and plant breeding
What is the graphic output from automated sequencing called?
a chromatogram
whole genome sequencing, exome sequencing, and targeted sequencing are all applications of what?
NGS
these are hundreds to thousands of different DNA sequences spotted onto glass microscope slide
DNA microarrays
What are DNA microarrays (gene chips) used for?
comparing base-pairing of two different sets of expressed gene sequences to the the template DNA microarray
In regards to resolving power, what things can be seen in a electron microscope? a light microscope? naked eye?
electron microscope: everything
light microscope: virus ribosome, bacterium animal cell, plant cell frog egg
naked eye: frog egg
Bright field is a type of light microscopy, how does it work?
light is transmitted through specimen
Phase contrast is a type of light microscopy, how does it work?
alterations in light transmitted as changes in brightness
How does sectioning tissue work?
very thin slices taken form embedded or frozen tissue
In regards to hematoxylin and eosin tissue staining, what is one tissue that is stained?
plant root
excitation and emission of wavelengths differ in ___ microscopy
fluorescence
In regards to fluorescence microscopy, there is more energy (excitement) at the lower or higher wavelengths?
lower; energy highest at violet, decreases to red
For each color, what is the associated wavelength?
Red: 660
orange: 620
yellow: 580
green: 540
blue: 480
violet: 420
What is the difference between decovolution and confocal imaging?
decovolution: removes light above and below focal plane of interest, mathematical analysis
confocal: focuses light only on the plane of interest, physical illumination
This shoots electron beams through magnetic coils
electron microscopy
What is the immunogold technique of electron microscopy?
antibody conjugated to a gold particle
What is siRNA and how is it involved in gene silencing?
small interfering RNA, is an intermediate in the RNA interference pathway; it occurs naturally in the cytoplasm or may be synthesized externally and introduced to the cell
This type of RNA is also involved in gene silencing and is the most widely used form of ddRNAi. It is produced inside the target cell from a DNA construct that has been delivered to the nucleus
shRNA (Short hairpin RNA)
