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 complementary 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

end labeling

adding probes to end

strand synthesis

incorporate label by synthesis; make new copy and add labels

fluorophores and haptens

non radioactive labels

southern blotting

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

use UV light to cross link DNA to membrane

fixation in southern, northern, and dot blotting

pre hybridization

block non specific binding sites in 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