Unit 12: In situ hybridization

What does in situ mean?

in the original position

What is in situ hybridization?

using a complementary labeled DNA or RNA probe to localize a specific DNA or RNA sequence in tissue

What is composed of a base, a sugar, and a phosphate group?

nucleotide

What is a base plus a sugar, no phosphate group?

nucleoside

Is adenine (A) double or single ring base?

double

Is guanine (G) double or single ring base??

double

Is cytosine (C) double or single ring base?

single

Is thymine (T) double or single ring base?

single

The end with the hydroxyl attached to the 3’ carbon is called the

3’ end of the DNA

The end with the phosphate attached to the 5’ carbon is called the

5’ end of the DNA

What is in situ hybridization?

localizing cytoplasmic mRNA or nuclear DNA by hybridizing a specific nucleotide sequence within cells and tissue to a complimentary strand of nucleotide probe

First step of ISH

pretreatment/prehybridization

Purpose of pretreatment/prehybridization

to reduce background staining

Second step of ISH

digestion

What does digestion do?

allows probes to penetrate tissue

Third step of ISH

denaturation

What is denaturation?

dissociation of two complimentary nucleic acid strands by breaking the hydrogen bonds between base pairs

Amount of heat dependent on type of ..

hydrogen bonds in the DNA helix

T/F denaturation can be done by a high (alkaline) pH

true

Fourth step of ISH

probe application

What is a probe?

a complimentary sequence of nucleotide bases specific to RNA or DNA sequence of interest

Fifth step of ISH

hybridization

What is hybridization?

the process of joining two complementary strands of DNA or one each of DNA and RNA to form a double-stranded molecule

What is the opposite of denaturation?

hybridization

What is accomplished by lowering the temperature or lowering the pH?

hybridization

__ need to be denatured prior to hybridization

double-stranded DNA and probes

Complementary sequence of bases to 5’AAGTCG3’?

5’CGACTT3’

Transcription is:

synthesis of complementary mRNA

What does ISH demonstrate?

DNA or RNA sequences of genes

With ISH, what’s the label attached to?

the complementary probe

What does C in CISH stand for?

chromogenic

How are complementary DNA strands arranged?

antiparallel

Genetic instructions are dictated by the order of…

nitrogen base pairs

Where does translation take place?

cytoplasm

Where does transcription take place?

nucleus

T/F viruses can be demonstrated with ISH

true

T/F if sequence of DNA nitrogenous bases is known for one strand, then the sequence of the other strand is also known

true

What can you do to increase stringency?

decrease salt concentration, increase formamide

What happens to Tm as homology increases?

Tm increases

Which of the following has the lowest Tm?

• 50% homology

• 75% homology

• 100% homology

50% homology

What does increasing stringency allow for?

more specificity, more homology

Which method can detect silent mutations?

ISH

Which base pair has the highest Tm?

G-C

Identify the hybrid

• patient DNA + probe

• probe + patient DNA

Another term for hybridization

annealing

If the probe is labeled with biotin, what detection system is used?

LSAB