lecture 1. 7/18 Flashcards
what dna technology rely on?
hybridization
hybidization assays main principle?
to dectect a dna molecule (target) within a complex mixture.
what is a probe?
dna sequence of knwon sequence. could be a dna sequence of a mutated gene.
why are probes labeled?
to be able to see. usually radioactive
main thing a probe must have?
complementary to the target sequence
what are the 3 possible outcomes of probing with target dna?
- target dna ca recombine
- probe can rennealed
- probe-target heteroduplexes. (best scenario)
stringency?
the degree to which non-complementary sequences are tolerated during hybridization.
the higher the stringency? what happens?
the fewer mismatches will be tolerated
what are the hybridization of the target gene and probe an example of?
orthologs or paralogs.
factors that correlates with stringency or decrease it?
salt concentration,
temperature
denature agents
different ways to make hybridization?
dna probe with dna target
dna probe with rna targe
rna probe with rna targets
Northern blotting mainly used for what?
to measure gene expression. look at rna sequences
western blotting used for what
to id proteins with antibodies after electrophoresis and electro-transfer. not used for diagnosis.
why to diagnosis is preferable to look into dna and not proteins?
hard to analyse proteins than it is to analyse dna. proteins can be sequenced but it is much harder. dna is much easier to do.
practical reason: much easier to get a dna sample because it can be collected anywhere in the body. to get a protein sample need to look at specific tissue that expresses it like the brain for huntingtons disease.
how southern blotting works?
target will be genomic dna (chormosomes)
not the whole chromosome, digest it with restrictive enzymes.
separate thorugh gel electrophoresis.
take a section of a similar size section from the gel and then do the hybridization with the probes.
Main feature of Southern: transfer dna to membrane.
put membrane in a bag, add buffer and probe, set temp. and allow hybridization.
wash membrane, take memebrane to x-ray and expose to film and develop it.
what the bands on the southern membrane represent?
correspond to size of segments.
would you expect to see differences between tissues in southern blots from a single patients?
no, bc it is genomic dnd, wont change from tissue to tissue
*in northern blotting (protein analysis) difference would be seen from tissues to tissues
polymorphism?
difference in sequences of alleles common in the population
what is restriction fragment length polymorphism? RFLP
a polymorphosm that results in change in the size of a restriction fragment
what are RFLP mainly used for?
look at different alleles
what type of sequence is used to id different alleles?
restriction site where restriction sites can cut
what is the meaning of the place where the probe binds?
where you can see in the southern blot
what is the banding pattern indicate in the southern blot?
different alleles both bind to the different blotting
what is the marker for the mutation on the allele?
the ??????
