1-37 Technology II Flashcards Preview

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Flashcards in 1-37 Technology II Deck (66):
1

Fish is used to...

benefit?

detect physical location of gene on intact chromosome.

in situ means "in place"
allows for evaluation of dividing and non dividing cells, while karyotyping requires dividing cells

2

steps of fish

1. cells that have been arrested in metaphase are treated to make them swell
2. then fixed onto surface of slide along with their chromosomes
3. slide is treated so chromosomal DNA is denatured into single strands
4. special DNA probes (small peices of ssDNA with a sequence from gene of interest that are flourescently labeled or are able to accept flourescent labeling) are flooded onto slide
5. probes hybridize only with complementary sequence - will be at site of gene of interest at particular chromosome
6. excess probes washed away
7. the probes are flourescently labeled if not already, excess washed away
8. viewed wtih flourescent microscope to reveal location of gene of interest

3

nucleic acid hybridization

forms basis of?

detects complementary nucleic acid sequences in complex mixtures.

many methods which determine size, quantity, and determine prescence of specific gene sequences

4

nucleic acid hybridization

1. high temp, dsDNA denatures to ssDNA
2. temp lowered, and if strands are complementary from other sources, will aneal - hybridization
3. regions of dsDNA where the ss anneal to each other are said to be homologous
4. high specificity of base pairing of compliemntary strands can be used to locate a specific nucleotide sequence in a sample
5. if DNA from one source is immobilized onto solid surface like nitrocellulose, homologous DNA from another source will hybridize with the immobilized strand - basis for various DNA probe techniques

5

basis for various DNA probe techniques?

if DNA from one source is immobilized onto solid surface like nitrocellulose, homologous DNA from another source will hybridize with the immobilized strand - basis for various DNA probe techniques

6

Gel electrophoresis is used to

forms basis of...

separate nucleic acid molecules and proteins by size

nothern, southern, and western blot

7

PCR requires

2 primers (ss complementary to template DNA), dNTPs, starting sample that contains template, a thermophilic polymerase

8

DNA electrophoresis allows us to determine

presence, size, and quantity of purified DNA

9

Southern Blot/Assay allows us to determine

presence and size of a specific DNA sequence in a complex mixture

dna/down/south

10

PCR does what?

amplifies a specific DNA sequence from a complex mixture

11

rtPCR

reverse transcriptase PCR - amplify a specific RNA sequence from a complex mixture

12

Multi-plex PCR

PCR done with multiple different primers, done clinically to screen for multiple alleles at once. analyze multiple sequences at once

13

Northern Blot/Assay allows us to determine

prescence and size fo a specific RNA sequence in a complex mixture/sample

NoRth - RNA

14

non-denaturing/Native protein Gel elecrophoresis

separate proteins based on size/shape/charge at a particular pH

– Separates based on charge. Used to distinguish between two different forms of the same protein with different charges due to amino acid changes.

15

Iso-electric focusing

separates proteins based on pI (pH at which protein is uncharged)

Another method for separation based on charge. Proteins are run along a gel with a stable pH gradient, and travel until they reach their isoelectric point.

16

Western Blot/Assay allows us to determine

presence, size, and abundance of a specific PROTEIN in a complex mixture/sample

17

Immuno-fluorescence microscopy

the presence and localization of a protein in a fixed tissue/cell sample

18

GFP florescence

– GFP absorbs light in the UV range, and emits light in the visible range. The gene for this protein can be fused to genes of interest, allowing protein dynamics to be followed in real time by looking for the fluorescence. Variants of this gene have been developed to fluoresce different colors, allowing researchers to follow a variety of proteins simultaneously.

19

Micro-array based expression analysis

a collection of microscopic DNA spots attached to a solid surface. They are used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

20

RNAi

main advantage?

A small sequence of double-stranded RNA (dsRNA) into a cell. An RNA endonuclease called a dicer cuts the dsRNA into small, single-stranded pieces, which are then directed to complementary mRNA sequences in the cell. Annealing to the mRNA causes it to get degraded by the dicer.

is that a natural system of gene expression is being harnessed.

21

Southern Blot

info given

steps(5)

Southern blot - detects specific DNA sequences in DNA samples

1. separate DNA samples based on size (use restriction enzymes then gel electrophoresis)
2. transfer to nitrocellulose sheet
3. to ID which band contains gene of interest (knowing sequence), build DNA probe with complementary sequence with radiolabel.
4. Treat nitrocellulose paper, labeled probe will hybridize with fragment of interest to form dsDNA
5. xray radiography to find where DNA probe is. Can go back to gel and further purify

22

Gel electrophoresis

info provided?

steps?

agarose used for?

SDS page

general method to separate nucleic acid molecules (DNA/RNA) and proteins by size

1. DNA with uniform - charge is loaded at cathode (-) and travels towards anode (+)
2. dna samples separate based on size, smaller moving further due to resistance of movement by gel

agarose used to separate large DNA fragments

SDS page - used to separate smaller peices of DNA or proteins - smaller pores in gel

23

mayn nucelic acid detection methods are limited by?

the quantities of DNA that can be isolated and manipulated

24

PCR is simply...

what are the boundaries/size of teh template determined by?

repeated rounds of primer detected DNA replication off of a rare tempalte, each round creating more and more.

determined by choice of primers

25

key to PCR?

use DNA polymerases from thermophilic (heat loving) microorganisms

26

describe the primers added in PCR

one is complementary to a region to the right on the top strand of DNA, one complementary to the left region of teh bottom strand

27

in what direction does the polymerase extend?

5--3

28

what is real time PCR?

machine uses flourescence to quantify product production at each step of teh reaction. the resulting curve of product vs time (cycles) can be used to accurately compare template abundance in different samples

29

in general, how is a multiplex reaction run?

special consideration?

multi plex PCR - single sample is simulatenously analyzed with multiple sets of primers added to the same PCR reaction

designed so that all products can be separated via gel electrophoresis

30

how can multiplex PCR be used to diagnose DMD?

DMD frequently caused by deletions of one or more exons for dystrophin gene.

-to analyze for exon deletions, multi-plex PCR is done with primers designed to amplify subsets of the dystrophin gene exons.

-you then run on gel electrophoresis against a standard "normal", adn see what exons are missing

31

why use PCR/gel electrophoresis to test for HIV?

how?

because its so sensitive that it can detect the pathogen before symptoms even start occuring - the best time to start treatment
"RT-PCR)
1. take blood from patient
2. centrifuge out infected plasma
3. extract RNA
4. use reverse transcriptase and PCR to amplify HIV cDNA
5. run on gel against controls (non infected)

32

to be able to clone a DNA insert into a cloning or expression vector...

both the DNA and cloning/expression vector must be digested with 2 restriction enzymes that create compatible ends (sticky being the best to ensure insert is incorporated at desired orientation)

33

cloning by PCR

fragments vs just the coding region

1. fragments of DNA can be amplified by PCR

2. to clone just the coding region (as introns have been spliced out) - isolate mRNA and us RT to make a DNA copy of the mRNA corresponding to the gene of interest prior to PCR amplification

34

to separate proteins based on size

differnet proteins have different charges based on their aa composition.

must make charges irrelevant - do this by heating in presence of detergent SDS. coats the protein in - charge.

35

denaturing protein electrophoresis

used to separate proteins based on size alone
-denature with heat and SDS that gives - charge
-use reducing agent (beta mercaptoethanol) to reduce disulfide bons

size alone.

36

what is non-denaturing protein electrophoresis? used for?

whats to compare changes in charge between two proteins (for example, a mutation in gene that causes genetic disease)

-leave out the step that involves heating, SDS, and mercaptoethanol

-protein separation will be based solely on charge and shape of protein

37

2D gel electrophoresis

1. IF in tube gel. tube is then placed on top of standard denaturing gel.
2. Gel electrophoresis is then run

compares proteins based on isoelectric point and size

38

DNA micro arrays

a collection of microscopic DNA spots attached to a solid surface. They are used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

39

microarray steps?

-allows comparieson in expression levels for all the geenes tested between two samples.
-RNA is isolated from reference sample and tagged with one color
-RNA is isolated from patient and tagged with another color
-two labeled samples are then mixed and hybridized to a single DNA-micro array slide.
-Flourecence in both colors is read by a microscope and the ratio between the two colors is caculated for each spot on the array.
-the ragio for each gene is reflection on the relative expression levels of each gene in the two samples

-ratio = 1, then the expression of the gene did not change between the reference and the patient sample

40

DNA microarray data is often depicted as

cluster analysis

41

what is cluster analysis

depection of DNA microarray data.
-data is compared to find those genes that are similarly expressed and whose expression changes in a similar manner under different conditions

-in this way, sets of genes that are coordinatedly regulated fall into "clusters"

Grouping of sets of genes (information obtained from DNA microarrays) in such a way that sets of genes in the same group (called a cluster) are more similar to each other than to those in other clusters.

42

genes found in the same cluster

frequently function in teh same pathway or cellular process. Ex) fibroblasts that were starved then fed serum. cluster analysis showed which genes were activated similarly to wound healing, cell cycle, and cholesterol biosynthesis. - serves as a way to hypothesize the activity of previously unknown genes

43

lung cluster example

adeno was split into 3 groups by the cluster analysis of teh micro array. These 3 groups were indistinguishable by pathologists. however, the cluster analysis showed that the closer to normal, the better the prognosis of the disease. The further form normal, and the more the adeno group was clustered with very serious lung diseases, the worse the prognosis.

Treatments can also be tailored to these groups

44

what do restriction enzymes do?

used to uniquely...

bacterial enzymes used to cut DNA molecules at specific recognition sequences.

uniquely characterize DNA molecules and in DNA cloning by recognizing palindromic sequences (nucleic acid sequence on ds DNA or RNA wherin treading 5-3 forward on one start matches the sequence reading backwards 5-3 on the complementary strand)

5-GAATTC-3
3-CTTAAG-5

https://www.youtube.com/watch?v=U2cKywEn6KY

45

restriction enzymes recognize

palindromic sequences that are not methylated (not self)

will result in two strands

5-GAATTC-5
3-CTTAAG-5

turns to

5-G AATTC-3
3-CTTAA G-5

forms sticky ends.

46

cloning in a plasmid vector

method used to isolate and amplify in bacteria a specific gene of interest
1. isolate particular sequence of dna
2. insert the fragment+abx resistance gene into a vector that can replicate in a host cell (use restriction same vector to cleave host as well as DNA fragment at the same palindromic sequences to create complementary areas
3. sticky ends of foriegn and plasmid hydridize and then sealed by ligase to form recombinant plasmid
4. plasmid introduced into bacteria via transformation
5. bacteria introduced to antibiotic
6. only bacteria which took up the recombinant plasmid will survive
7. replication origin allows cell to replicate using teh host replication enzymes

47

3 elements of a fragment that can be inserted into a vector

1. cloning site of host where fragment can be inserted
2. drug resistant gene within fragment
3. reaplciation origin allowing plasmid to replicate in host cell

48

Cloning via lambda bacteriophage

type of virus that infects e.coli. better for efficiently cloning larger segments of DNA

We want to replace DNA molecule in lambda phage virus with DNA of interest of appropriate size
1. extract phage DNA and use restriction enzymes, ligate target DNA into lambda DNA to produce recombinant lambda DNA, put back in phage
2. expose e.coli cell to lambda phage
3. lambda phage will infect and undergo lysogenic (passive) phase that will replicate along iwth the e.coli for many rounds of replication
4. extract replicated DNA from E.coli

49

cloning via cosmid vector

advantage?

1 - clone DNA into vector as you would any other plasmid
2 - introduce DNA into bacterial cell via phage
3. propogate as a plasmid

clone even larger peices of DNA

50

requirments of plasmids/cosmids

have origin of replication
have selectable marker
cloning site

cosmid must also have a cos site

51

purpose of a COS site?

cos site allows DNA to be packaged into phage particles

52

RNAi

double stranded RNA molecules corresponding to a particular gene could be used to down-regulate the expression of that gene

-mechanism by which cells protect themselves against RNA viruses and rampant retro-transposon amplification

53

RNAi as treatment?

RNAi has been shown to downregulate the expression of genes in human cells - emerging techn could be used to treat diseases caused by inappropriate gene expression or expression of damaging mutatnt alleles.

54

Lentivirus delivery system of shRHAs

in humans must introduce genes encoding for both strands of RNA to initiate RNAi.

-construct gene that will express a self complimentary RNA that will form a hairpin
-hairpin introduced via viral/plasmid vectors
-dsRNA enters cell, recognized by RNA Endonuclease "DICER" with helicse
-dicer cuts dsRNA into 23 peices that then direct, via homolgy, the complex to a complementary mRNA species
-Dicer then cuts the mRNA leaving it succeptible for destruction by cellular exonucleases.

55

Western Blotting Steps

1. We have sample of proteins within a cell. Protein we want is within cell.
2. Centrifuge to isolate protein samples
3. Gel electrophoresis (SDS polyacrylamide)
4. Transfer to polymer sheet
5. Synthesize monoclonal antibody that can bind to the antigen (protein) that we want to locate
6. Mix sheet with antibody mixture
7. Antibodies will bind to one of the bands
8. Synthesize an antibody to the antibody (Secondary antibody) with a radioactive label
9. Expose to x-rays

56

Southern Blotting

1. take dsDNA with gene of interest, know sequence
2. Expose to specific restriction enzymes, one of the fragments will carry gene of interest
3. Place in solution and denature with heat - left with single stranded DNA molecules
4. Place into gel electrophoresis (agarose if a large fragment), separated based on size
5. Transfer gel to nitrocellulose sheet. Which band has the strand of interest?
6. Build a DNA probe with complementary nucleotide sequence to that fragment that we want to isolate and radioactively label.
7. Use x-ray radiography to detect label

57

Northern Blotting

Southern blotting but with RNA molecules, use an RNA probe instead

58

pyrosequencing

produces light when nucleotide encorporated and sequence extended. Nucleotides are dispensed one at a time in a defined order. Excess are removed before dispensing next one. If the complement of the nucleotide dispensed is present int eh template, light is produced. Light produced is converted into a peak (pyrogram). Height = proportional to number of nucleotides incorporated.

59

during illumina sequencing..

DNA sequence is analyzed base by base,making it highly accurate

Sequence generated can then be aligned to reference sequence that looks for matches or changes in sequenced DNA

60

2 dimensional electrophoresis

IEF followed by denaturing gel electrophoresis. This technique has high enough resolving power to describe the entire proteome of a cell or tissue.

61

Allows clinicians to screen for specific alleles. It also has particularly useful application in detecting minute amounts of HIV nucleic acids in blood samples.

PCR

62

(2)– Can be used to distinguish variable forms of proteins from one another, like normal hemoglobin vs sickle cell hemoglobin.

Non denaturing protein electrophoresis and IEF

63

Allows clinicians to determine the nature of the kinds of proteins being synthesized in a particular cell or tissue.

2d gel electrophoresis

64

Allows clinicians to determine the expression level of very many genes at once in a particular cell or tissue.

DNA Microarrays and Cluster Analysis

65

Has the promising application of allowing clinicians to directly influence gene expression at the level of translation.

RNAi

66

Allows researchers to learn more about protein expression patterns in developing and adult organisms alike.

following protein expression using GFP

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