L6 Hybridization

Question 1

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

Answer 1

Blotting/Hybridization Techniques

Question 2

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

Answer 2

Northern Blot

Question 3

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

Answer 3

Southern Blot

Question 4

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

Answer 4

Western Blot

Question 5

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

Annealing/hybridization

Answer 5

Probes (detection with probes)

Question 6

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

Answer 6

Annealing/hybridization

Question 7

• Radioactive (P32) label
• Chemiluminescent compound
• Fluorescent compound
• Enzymatic label

Answer 7

ID of target detected/identified by labeling probes with

Question 8

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

Answer 8

nitrocellulose paper

Question 9

Separate DNA fragments by ___ and ___ ________.

Answer 9

RE, gel electrophoresis

Question 10

ASO (stands for?)

Answer 10

Allele-specific Oligonucleotide

Question 11

How many probes does ASO have for each allele?

Answer 11

2

Question 12

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

Answer 12

phenylalanine 508

Question 13

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

Answer 13

ASO for Cystic Fibrosis Detection

Question 14

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

Answer 14

Capture hybrids

Question 15

• DNA is denatured
• Hybridized to an RNA probe
• Captured by bound anti-DNA/RNA antibodies

Answer 15

Chemiluminescence detection of hybrid molecules (DNA/RNA)

Question 16

PCR steps

Answer 16

• Denaturation
• Annealing
• Extension (elongation)

Question 17

Denaturation temperature

Answer 17

94

Question 18

Extension (elongation) temperature

Answer 18

72

Question 19

Taq polymerasae

Answer 19

Thermophilus aquaticus

Question 20

Termination temperature

Answer 20

4

Question 21

Repeat for ___ - ____ cycles

Answer 21

2 - 30

Question 22

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

Answer 22

T. aquaticus, hot springs

Question 23

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

Answer 23

75 - 80, 150

Question 24

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

Answer 24

RT-PCR

Question 25

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

Answer 25

quality and purity

Question 26

3 enzymes for RT-PCR

Answer 26

Reverse transcriptase RNAse H Taq DNA polymerase

Question 27

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

Answer 27

NASBA and TMA Isothermal

Question 28

Hybrid capture bDNA

Answer 28

Signal Amplification Methods

Question 29

Anti-DNA-RNA hybrid antibody

Answer 29

Hybrid capture

Question 30

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

Answer 30

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

Question 31

Sequencing method of choice

Answer 31

Sanger method of choice

Question 32

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

Answer 32

Sanger

Question 33

DNA sample divided into 4 separate reactions (tubes)

Answer 33

Sanger method

Question 34

______ will prevent addition of further nucleotides

Answer 34

ddNTPs

Question 35

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

Answer 35

ddGTP ddATP ddCTP ddTTP

Question 36

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

Answer 36

Next Generation of Sequencing (NGS)

Question 37

- 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

Answer 37

Basic major procedure of next generation sequencing

Question 38

powered by semiconductor chips technology

Answer 38

Ion torrent

Question 39

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

Answer 39

Illumina (SOLEXA) sequencing

Question 40

- 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

Answer 40

Bridge Amplification - Illumina SBS technology

Question 41

Use Allylamine-dUTP for

Answer 41

FISH DNA Probe Labeling

Question 42

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

Answer 42

colcemid

Question 43

Burst cell and load cells onto ____ ______ - FISH technique

Answer 43

glass slide

Question 44

- 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

Answer 44

FISH Clinical Utility

Question 45

P24

Answer 45

Gag

Question 46

Reverse transcriptase, integrate, protease

Answer 46

Pol

Question 47

Envelope components; gp120, gp41

Answer 47

Env