Flashcards in Lecture 6 Part 1 Deck (42)
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1
Polymerase Chain Reaction (PCR)
-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:
1. primers
2. DNA polymerase
3. dNTPS- deoxynucleotides
4. thermal cycling - a series of temperature cycles
2
primers
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
PCR steps
-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
Endpoint - PCR
-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
gene expression experiments typically measure ____ rather than RNA
cDNA = complimentary DNA
6
reverse transcription PCR (RT-PCR)
-measures fluoresce and amplification in real time
PCR ---> cDNA ---> + master mix + primers ---> real time qPCR machine
7
Real time quantitative PCR
-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
how does real-time quantitative PCR work?
-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
why is it called Real- Time?
• 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
cycle threshold Value (Ct)
-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
housekeeping genes (control)
-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
microarray
-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
sequencing techniques
-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
T or F: DNA methylation does not interfere with complimentary base pairing
true
15
Mapping DNA Methylation 3 methods
-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
chemical conversion of unmethylated cytosine
-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
Histones are what?
proteins
18
proteins can be detected by what?
-immunohistochemistry
-using antibodies to detect antigens (proteins) in tissues or cells
19
immunohistochemical technique steps
-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
the technique commonly used to identify the presence or absence of a histone modification in cells/tissues is called?
western blotting
-used to quantify or measure a protein
21
Western blot
-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
size-based separation of proteins
proteins will move in a different speed depending on how big they are
23
western immunoblot
-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
western blot advantages
-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
