Hybridization Formats

Question 1

What are the 3 hybridization formats?

Answer 1

solution - homogenous
solid - mixed phase
in-situ

Question 2

Components of mixed phase system and function?

Answer 2

solid transport - target DNA immobilized

solution - probe

Question 3

solid support types?

Answer 3

Nitrocellulose
latex beads
magnetic beads

Question 4

Explain the process of dot blot method

Answer 4

denaturation
immobilization
pre-hybridization
probes are added
washing
xray expose

Question 5

Why the filter is pre-hybridized?

Answer 5

to -bg noise. omit non specific binding sites

Question 6

How and what stringency is applied?

Answer 6

high

Question 7

What may be the reasons for bg noise?

Answer 7

bad washing

inefficient blocking

Question 8

Why southern blotting is used?

Answer 8

To detect specific dna sequences

Question 9

Explain the process of southern blotting.

Answer 9

restriction digestion
agarose gel electro.
blot on a gel on a sponge - buffer move by capillary
membrane is separated & baked
prehybridization
probs adding
xray

Question 10

What to do to hybridize RNA? What is done differently in n. blotting?

Answer 10

formaldehyde in gel - resolve 2ry str.s.

diazobenzyloxymethyl ppr

Question 11

What are the alternatives for southern blotting?

Answer 11

microtiter wells for immobilization

hybridization in dried agarose gel

Question 12

What are the applications of solid hybridization?

Answer 12

genome analysis
gene discovery & mapping
diagnostics
forensics

Question 13

What are the advantage and limitation of solution hybridization?

How come sol. hybri. is the fastest?

Answer 13

• fastest rate
• difficult to separate

-probe finding target chance is high

Question 14

Explain the process of solution hybridization?

Answer 14

hybridization
HAP added & mixed well - HAP binds to ds DNA
cetrifugation
wash the pellet
quantification - colorimetry / scintillation counter

Question 15

In situ hybridization

For what it is carried out?

Answer 15

• to identify seq.s in the cells/tissues themselves

Question 16

In-situ hybridization

how specimens are kept?

Answer 16

fixed on micro. slides - formalin fixed paraffin embedded tissues

Question 17

In-situ hybridization

What are the solid support and the solution?

Answer 17

• cells

- probe

Question 18

In-situ hybridization

What are the uses?

Answer 18

• Histology & cytology labs
• detection of viral seq.s - their intracellular location & no. of viral seq.s per cell
• diagnostics - detect chromo. translocations, deletions, cancer, FISH

-Research - localizing genes and their expression, -mapping genes

Question 19

Chromosome In-situ hybridization

What are the uses?

Answer 19

• cytogenetic analysis

- diagnose chromo. abnormalities

Question 20

Explain the process of chromo. In-situ hybridization? And as to why each is added.

Answer 20

• micro. slides of cells in meta/prometaphase prepared(why)
• treated with RNAse & proteinase K(why)
• denatured by formamide
• hybridized with probes
• washing
• Detection using FISH using a fluorescent microscope

Question 21

How to use tissues in In-situ hybridization?

Answer 21

• paraffin removed
• mounted on a micro. slide
• hybridized with probes - cDNA or RNA(higher sensivity but unstable)
• Do FISH

Question 22

How to prepare a blood culture for FISH?

Answer 22

ss