Lecture 6 Part 1 Flashcards Preview

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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

genetic engineering is used to direct what?

gene expression