L6 Hybridization Flashcards

1
Q

Using specific probes that are labelled specific sequences of DNA can be identified

A

Blotting/Hybridization Techniques

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Transfer of an RNA sample separated and identified using DNA or RNA probes

A

Northern Blot

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Transfer of a DNA sample separated and identified using DNA or RNA probes

A

Southern Blot

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Transfer of a protein sample separated and identified typically using an antibody

A

Western Blot

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

ssDNA that will base pair with a complementary sequence of either RNA or DNA

Annealing/hybridization

A

Probes (detection with probes)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Stringency: heat, chemical, salt affect binding sensitivity and specificity

A

Annealing/hybridization

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q
  • Radioactive (P32) label
  • Chemiluminescent compound
  • Fluorescent compound
  • Enzymatic label
A

ID of target detected/identified by labeling probes with

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Separated Nucleic Acids are blotted onto _____ ______ by suction of buffer through gel and paper

A

nitrocellulose paper

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Separate DNA fragments by ___ and ___ ________.

A

RE, gel electrophoresis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

ASO (stands for?)

A

Allele-specific Oligonucleotide

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How many probes does ASO have for each allele?

A

2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Cystic fibrosis - loss of ____________ leads to one form of the disease

A

phenylalanine 508

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Probes
- AsOn = WT (wild type)
- ASOx = mutant cystic fibrosis gene (loss of phenylalanine 508)

A

ASO for Cystic Fibrosis Detection

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

RNA:DNA hybrids are captured onto a microtiter well coated with capture antibodies specific for RNA:DNA hybrids

A

Capture hybrids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q
  • DNA is denatured
  • Hybridized to an RNA probe
  • Captured by bound anti-DNA/RNA antibodies
A

Chemiluminescence detection of hybrid molecules (DNA/RNA)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

PCR steps

A
  • Denaturation
  • Annealing
  • Extension (elongation)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Denaturation temperature

A

94

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Extension (elongation) temperature

A

72

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Taq polymerasae

A

Thermophilus aquaticus

20
Q

Termination temperature

A

4

21
Q

Repeat for ___ - ____ cycles

A

2 - 30

22
Q

Taq polymerase isolated from bacterium ________ that lives in ____ ______ and hydrothermal vents

A

T. aquaticus, hot springs

23
Q

Taq optimum temp ______ for activity polymerization at a rate of about ____ nucleotides per second per enzyme molecule

A

75 - 80, 150

24
Q

produce a DNA copy of the RNA template using either random primers, an oligo(dT) primer or sequence-specific primers

A

RT-PCR

25
Q

The _____ ______of the RNA template are crucial to the success of RT-PCR

A

quality and purity

26
Q

3 enzymes for RT-PCR

A

Reverse transcriptase
RNAse H
Taq DNA polymerase

27
Q

More clinically used because it is isothermal and begins w. RNA

A

NASBA and TMA Isothermal

28
Q

Hybrid capture
bDNA

A

Signal Amplification Methods

29
Q

Anti-DNA-RNA hybrid antibody

A

Hybrid capture

30
Q

More recent efforts have focused on the development of bDNA assays for the quantification

A

HIV-1 and hepatitis C virus (HCV) RNA,

31
Q

Sequencing method of choice

A

Sanger method of choice

32
Q

Requires ssDNA template, DNA primer, DNA polymerase, labeled nucleotides and modified nucleotides to terminate DNA elongation

A

Sanger

33
Q

DNA sample divided into 4 separate reactions (tubes)

A

Sanger method

34
Q

______ will prevent addition of further nucleotides

A

ddNTPs

35
Q

4 tubes in Sanger
G =
A =
C =
T =

A

ddGTP
ddATP
ddCTP
ddTTP

36
Q

Ultra-high throughput sequencing
The whole human genome (as many as 3 billions NA) with (Illumina) may be run in parallel in 30 hours

A

Next Generation of Sequencing (NGS)

37
Q
  • Fragmentation of DNA
  • Adapter are ligated
  • Denature to single strands
  • Formation of clonal cluster or bead populations
  • PCR amplifies DNA strands on flow cell or beads
A

Basic major procedure of next generation sequencing

38
Q

powered by semiconductor chips technology

A

Ion torrent

39
Q

based on sequencing-by-synthesis operations and reversible dye-terminators

A

Illumina (SOLEXA) sequencing

40
Q
  • Add dNTPs, and DNA polymerase enzyme to elongate DNA strands
  • Repeat until we have millions of dense clusters of DNA. The reverse strands are then cleaved and washed aways
A

Bridge Amplification - Illumina SBS technology

41
Q

Use Allylamine-dUTP for

A

FISH DNA Probe Labeling

42
Q

Harvest ______ (Will be stopped at metaphase) treated cultured cells in hypotonic solution → full cells – FISH technique

A

colcemid

43
Q

Burst cell and load cells onto ____ ______
- FISH technique

A

glass slide

44
Q
  • Used to identify the presence and location of a region of DNA or RNA (large fragment) within morphologically preserved chromosome preparations, fixed cells or tissue sections
  • This means you can view a segment or entire chromosome with your own eyes
  • Was often used during M phase but is now used on I phase chromosomes as well
A

FISH Clinical Utility

45
Q

P24

A

Gag

46
Q

Reverse transcriptase, integrate, protease

A

Pol

47
Q

Envelope components; gp120, gp41

A

Env