DNA HYBRIDIZATION

Hybridization

a phenomenon in which single stranded deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) molecules anneal to complementary DNA or RNA

hybridize

Lowering the surrounding temperature allows the single-stranded molecules to anneal or ”__________” to each other

DNA PROBE HYBRIDIZATION

generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences

genetic distance or degree of genetic similarity

DNA PROBE HYBRIDIZATION is usually used to determine the ____________ or _____________ between two organisms

gene probe

single stranded piece of DNA complementary to a desired target DNA sequence that is labelled

fluorescent dye or radioactive label

gene probe is usually labelled with _____________ or ____________

Nick translation, Random primed DNA labelling, End labelling of DNA, End labelling of RNA

methods in ASSAYS: PREPARATION OF NA PROBES

Nick translation

used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2)

3-hydroxyl groups

in nick translation, what is created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse)

deoxyribonuclease 1

in nick translation, Free 3-hydroxyl groups are created within the unlabeled DNA (nicks) by _____________

addition of dNTPs in 5’ to 3’ direction and removal of nucleotides in 5’ to 3’ direction

two main function of Polymerase 1

nucleotide is nicked by DNAse 1, polymerase 1 fills the gap with labelled nucleotide, moves in a 5’ to 3’ direction

illustrate NICK TRANSLATION

Klenow fragment of DNA Polymerase

what polymerase is used in Random primer labelling of DNA

it lacks 5’ to 3’ exonuclease activity

why is klenow fragment used in random primer labelling of DNA

single stranded DNA probe, anneal random primers to gene probe, Klenow DNA polymerase synthesize a complementary strand of DNA with labeled dNTP

illustrate random primed labelling of DNA

5’ or 3’ end labelling

Simplest form of labelling DNA

5’ end-labelling

involves a phosphate transfer or exchange reaction where the 5-phosphate of the DNA to be used as the probe is removed and in its place a labelled phosphate, usually 32P, is added

alkaline phosphatase

used to remove the existing phosphate group from the DNA

polynucleotide kinase

catalyzes the transfer of a phosphate group (32^P-labelled) to the 5’ end of the DNA.

purify gene probe fragment or synthesize oligonucleotide, alkaline phosphatase treatment of probe to remove 5’ phosphatase, polynucleotide kinase transfers phosphate group from donor to 5’ end of probe, 5’ end is radiolabeled and gene probe is purified

Illustrate end labelling at 5’ end

synthesize oligonucleotide or purify gene probe fragment, transfer labelled dNTP to the 3’ end using terminal transferase, 3’ end is radiolabelled and gene probe is purified

Illustrate end labelling at 3’ end

weak hydrogen bonds

The strands of DNA are linked together by ? when acted upon by denaturation

Denaturation, annealing, extension

steps in PCR/ principle in PCR

denaturation (95 degrees), annealing (40 degrees, primer anneal), extension (72 degrees, Pol I), dna replicated

illustrate PCR

DNA HYBRIDIZATION

the process of establishing a non-covalent, sequence-specific interaction between two or more complementary acids of nucleic acids into a single hybrid

duplex

two hybrid strands is referred to as a

bacterial taxonomists

DNA-DNA Hybridization values have been used by ___________ since 1960

To determine relatedness between strains and and due to declination of bacterial species

purpose of DNA hybridization

DNA probe to Hybridization to determining the degree of genetic similarity

illustrate Hybridization in bacterial taxonomy

organisms are identical

complete hybridization of two organism strand means

organisms are related

partial hybridization of two organism strand means

organisms unrelated

no hybridization of two organism strand means

1. heat to separate strands

2. combine single strands of DNA

3. cool to allow renaturation of double-stranded DNA

4. determine degree of hybridization

steps in dna hybridization two organisms dna for determining degree of genetic similarity

1. ds DNA bound to the column

2. the mixture is heated

3. mixture is heated in small steps

4. each step - column washed

5. sequence melt - ssDNA washed off

6. temp at which labelled DNA comes off the column reflects the amount of similarity between sequences

7. combined results - determine the degree of genetic similarity between organisms

access melting profile of hybridized DNA steps

Homologous hybrid

if 2 strands species DNA have a probe attached to it, what type of hybrid is it?

heterologous hybrid

if 1 strand species DNA have a probe attached to it and the other has none, what type of hybrid is it?

Homologous hybrid

if both strand species DNA have no probe attached to it, what type of hybrid is it?

DNA fragmented by bead beating

it ensures randomness in DNA microarray

Agarose gel

used to elute and purify DNA in DNA microarray

T3-T7 promoter primer

In DNA microarray, what is used to amplify PCR