L9 - molecular bio 3 - nucleic acid probes and hybridization Flashcards
what is nucleic acid hybridization
method to identify nucleic acid clones/fragments containing specific sequences
what can separate a DNA strand
heat
alkali conditions
define hybridize
complimentary strands will join up (hybridize)
can DNA and RNA hybridize to ssDNA
yes
why do we carry out nuclear acid hybridization technique?
may have a sequence of DNA and we want to see what plasmids in the cDNA have this sequence
how is the DNA probe made
for hybridization
- denature template and add primer
- add DNA pol and radiolabelled ddNTPs for it to incorporate along from the primer
- denature again to free the labelled primer
what is the name of the modified DNA pol used in process of making hybridization probe
klenow fragment
describe process of colony blot hybridization
- bacterial colonies (containing cDNA plasmids) grown then transferred onto DNA binding material
- bacteria lysed and denatured using alkali
- place in solution with labelled probe
- complimentary sequences will hybridize
- rinse and expose to x-ray film to identify radiolabels
- separate hybridised colonies and grow
example of DNA binding material
nylon
nitrocellulose
when is colony blot hybridization used
to compare probe to cDNA library
when is southern blot hybridization used
we have an isolated sequence of DNA
we want to see what other DNA contains this sequence
describe process of southern blot
- digest DNA with RE
- separate fragments by Gel electrophoresis
- denature fragments in alkali
- transfer to nitrocellulose/nylon
- add labelled primer in solution with the ssDNA fragments
- complimentary regions will hybridize and can be detected
how are the ssDNA transferred from the gel to the DNA binding membrane?
this part is the actual southern blot method
membrane placed on top of gel with paper towels on top of membrane
paper towels suck the alkali solution up onto the DNA binding membrane
when is northern blot used
when we have an isolated sequence of DNA
we want to see what RNA contains the sequence
eg to see if a gene (in DNA) is being expressed (in RNA)
describe process of northern blot
- mRNA extracted from sample and separated on agarose gel
- transferred straight to membrane (no need to denature as ss)
- labelled DNA probe added, will hybridize at complimentary sequences
(in a northern blot) if our DNA probe doesn’t hybridize to any of the RNA what does this mean?
the gene from DNA isnt being expressed
uses of northern blot
- can compare expression (and extent of expression) of gene between tissues
- can see how a disease affects gene expression
- can see how gene expression changes over time (development)
define in situ hybridization
hybridizing a probe directly to RNA without blotting onto membrane
can be carried out on tissue sections or entire organism
advantages of in situ hybridisation
can use labelled probe to identify cellular and tissue distribution of mRNA (of specific genes)
what are DNA microarrays
modern devices that use nucleic acid hybridization to rapidly measure which genes are present in a tissue sample (simultaneously)
study expression of a gene across entire genome
how do DNA microarrays work
uses reverse approach
- all mRNA converted to cDNA, fluorescently labelled and used as probe
- probe applied to array which contains spots where ssDNA from each gene in genome is laid out
- labelled cDNA probe will bind complimentarily and their fluorescence will be detected by chip reader
what does brightness of spots in a microarray indicate?
extent of expression of a gene
define transcriptome
set of transcripts in a cell/tissue/sample
define transcriptomics
the study of transcriptomes
what is comparative DNA microarrays
having 2+ samples, labelling different colours and hybridizing them to the same chip
eg sample 1 red
sample 2 green
genes expressed in both - yellow
genes expressed in only one sample : green/red
