Lecture 6 Part 1 Flashcards
(42 cards)
1
Q
Polymerase Chain Reaction (PCR)
A
- amplifies nucleic acids and allows us to quantify them so we can detect it
- specific sequences within DNA molecules can be identified by copying or amplifying the sequence of interest using:
- primers
- DNA polymerase
- dNTPS- deoxynucleotides
- thermal cycling - a series of temperature cycles
2
Q
primers
A
short segments of complimentary DNA that base-pair with the template DNA upstream of the region of interest and serve as recruitment sites for the polymerase
3
Q
PCR steps
A
- melting : heat up sample to separate DNA strands
- annealing : cool down sample to allow primers to base part to complementary DNA template
- extension: heat up sample so that DNA polymerase extends primer and creates new strand of DNA
- exponential amplification: process is repeated, and the region of interest is amplified (each time we amplify we double the material )
- up to 40 cycles
4
Q
Endpoint - PCR
A
- used to detect and quantified amplified sequences done by PCR
- amplified DNA is placed into gel near the cathode of an electrical field
- b/c of electrical charges DNA moves and migrates through the gel to anode and separates by size
- the bands can be visualized with ethidium bromide, which causes DNA to fluoresce
- good to identify presence vs. absence sequence
5
Q
gene expression experiments typically measure ____ rather than RNA
A
cDNA = complimentary DNA
6
Q
reverse transcription PCR (RT-PCR)
A
-measures fluoresce and amplification in real time
PCR —> cDNA —> + master mix + primers —> real time qPCR machine
7
Q
Real time quantitative PCR
A
- PCR product is measure at each cycle
- PCR product is measured via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules (amplicons) generated
- fluorescent reporters used in real time PCR include double-stranded DNA (dsDNA)
- binding dyes or dye molecules attached to PCR primers or probes that hybridize with PCR product during amplification
8
Q
how does real-time quantitative PCR work?
A
- a probe specific to the gene of interest is labeled with a reporter dye and a quencher dye
- when incorporated to PC product, the reporter is released
- primer and probe work together so that we wont see the probe light until after extension occurs and the reporter is rereleased
- every time an amplicon/ PCR occurs we get a fluorescent indicator light signaling it occur
9
Q
why is it called Real- Time?
A
- 0 -40 : Every time we go through a cycle fluorescence indicators increase making it visible around cycle 14
- There is fluorescence in all the samples
○ However if there is more DNA in sample = see fluorescence sooner
• Lower cycle values = more starting material
-Higher cycle values = lower starting material
10
Q
cycle threshold Value (Ct)
A
- the number cycles needed to reach a certain level of fluorescence
- the more copies of mRNA present, the lower the Ct
- early fluorescence means that they had high expression
- late fluorescence means lower expression
11
Q
housekeeping genes (control)
A
- used to account for technical variation
- pick a gene we don’t think should be change
- a negative control
- have no reason to believe that it should be more expressed or less expressed and use it to measure thee relative difference
12
Q
microarray
A
- thousands of primers/ probes hybridize to complementary sequences in sample thus allowing parallel analysis for gene expression and gene discovery
- single experiment can provide information on thousands of genes simultaneously
- using hundreds of primers and then picking a few of those to see whether they are involved in whatever it is I am interested in
13
Q
sequencing techniques
A
-actual sequences present in samples are “read”
- base pair resolution
- highly sensitive
- expensive
-actually allows you to assess the sequences that are present by reading the resolution and aligning it to a
14
Q
T or F: DNA methylation does not interfere with complimentary base pairing
A
true
15
Q
Mapping DNA Methylation 3 methods
A
- selective digestion of unmethylated DNA using restriction enzymes
- CONVERSION OF UNMETHYLATED CYTOSINE BY SODIUM BISULFITE TREATMENT
- selectively enriching locations containing methylation using 5mc antibodies
16
Q
chemical conversion of unmethylated cytosine
A
- bisulfite treatment introduces specific changes in the DNA sequence depending non the methylation status of individual cytosine residues
- treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected
- treat cytosine with bisulfite —> methylated cytosine stays but unmethylated cytosine changes to uracil
17
Q
Histones are what?
A
proteins
18
Q
proteins can be detected by what?
A
- immunohistochemistry
- using antibodies to detect antigens (proteins) in tissues or cells
19
Q
immunohistochemical technique steps
A
- isolating proteins in the cell/tissue of interest
- treating the sample with an antibody that will specifically recognize the protein of interest
- visualizing the antibody-protein complex to measure the protein of interest, Specific post-translational modifications of proteins can also be identified
20
Q
the technique commonly used to identify the presence or absence of a histone modification in cells/tissues is called?
A
western blotting
-used to quantify or measure a protein
21
Q
Western blot
A
- separate proteins by size using electrophoresis
- transfer proteins on to a membrane
- incubate membrane in primary antibody detecting protein of interest
- incubate in secondary antibody
- imaging
22
Q
size-based separation of proteins
A
proteins will move in a different speed depending on how big they are
23
Q
western immunoblot
A
- after proteins are separated on gel they must be transferred to a membrane where the protein of interest can be visualized
- membrane is treated with an antibody that will bind to target protein and a secondary antibody that will bind allow visual detection of the protein
24
Q
western blot advantages
A
- gold standard for looking at proteins
- can detect multiple forms of protein
- can determine size of detected protein
- used when people want to know what’s the actual quantity
25
western blot disadvantages
- low spatial resolution
- results can be inconsistent
- causes the loss of some proteins/sample during process
26
T or F: to determine the critical enzymatic events we manipulate the brain
true
27
methods for manipulating the brain
- brain lesions
- pharmacological agents
- molecular techniques
28
what are some pharmacological agents
- receptor agonists/ antagonists
| - HDAC inhibitor and DNMT inhibitor drugs
29
molecular techniques
- genetic engineering
- transgenic mice
- viral vector mediate gene transfer
- in vitro luciferase assays
30
genetic engineering is used to direct what?
gene expression
31
Cre-Lox technology is used to what?
manipulate genetic material in order to fain experimental control over gene expression
-used to control transcriptional
32
Cre-Lox steps
specific promoter ---> CRE recombinase ---> CRE controls the expression of a "floxed" gene ---> LOxP controls expression in gene X
33
transgenic mice pros
- can get very specific silencing of a specific gene
- new approaches allow more control over where and when gene is turned on/off
-non invasive control of gene expression
34
transgenic mice cons
-technology has been traditionally optimized for only mice
35
Cre-Lox system relies on two components to function ?
- a site specific recombinase called CRE
| - DNA sequence called LoxP
36
LoxP sites can be oriented in such a way that Cre recombinase can cause ?
- the deletion of a genetic segment
| - the expression of a genetic segment
37
viral vector mediate gene transfer
- used to affect a specific sequence
- control over where and when a gene is expressed or silenced
- genetically engineered viruses can be used to introduce DNA inside specific populations of cells
- can be introduced into a specific brain region at a specific window of development
- vectors expressing Creativity are often used with mouse models carrying a "floxed" gene
-vectors often coexpress a reporter such as green fluorescent protein to determine which regions are transfected by vector
38
luciferase reporter assay
- in vitro (cells in a dish)
- the transcriptional activity of a genetically engineered construct can be monitored using light (firefly luciferase enzyme gene)
- involves cloning your DNA sequence of interest upstream from the luciferase gene in viral expression vector
-produces an easy to light signal whenever transcription has occurred
39
chromatin immunoprecipitation goal
to isolate DNA bound to a specific protein of interest
40
ChIP is frequently used to determine
- transcription factor binding to promoter (which genes does a transcription factor regulate )
- acetylation status of promoter regions using antibodies for acetylated an non-acetylated histones
41
ChIP steps
- use freehand or punch sample of tissue
- sample is fixed, so that protein becomes cross-linked to DNA
- an antibody is used to "pull down" chromatin
- dissociate DNA from protein (reverse crosslink)
- analyze DNA using either PCR for sequencing
42
which method uses an antibody to locate a particular protein and gets rids of everything but the DNA
ChIP
