lecture 6-nucleic acid hybridization

nucleic acid hybridization

single stranded nucleic acid from different sources but complementary sequences form double stranded molecules

probes

a fragment of DNA or RNA of variable length which is used to detect the presence of homologous nucleotide sequences in a sample

PCR or DNA cloning

how do we get DNA probes

transcription of cloned DNA

how do we get riboprobes

chemical synthesis

how do we get oligonucleotide probes

add label to 5' end of probe

how do we label probes in end labeling

DNA or RNA template, DNA or RNA polymerase, and labeled nucleotide

how do we label probe in strand synthesis

fluorophores and haptens

non radioactive labels

biotin and digoxigenin

examples of haptens

southern blotting

uses DNA probe to detect a sequence in a DNA sample and identify the size of the fragment containing it

to fix DNA to membrane using cross linking

why do we use UV light in Southern blotting

northern blotting

uses a DNA or RNA probe to assess presence and quantity of mRNA in a sample

Dot blotting

detect and semi quantitate a DNA or mRNA sequence in a sample without electrophoresis

nylon

what membrane is used in dot blotting

checkboard DNA-DNA hybridization

detection and semi quantification of up to 40 bacterial species un up to 28 dental samples using whole genomic probes

in situ hybridization

uses a DNA or RNA probe, usually fluorescent, to detect and localize a DNA or mRNA in tissue or cell

DNA microarrays

a glass or silicon chip with thousands of unlabeled DNA probes spotted on it for hybridization with labeled nucleic acids from samples

human oral microbiome identification microarray

sample hybridized simultaneously agaist 300 different species per slide