Flashcards in ALL THE THINGS Deck (87)
What is a probe?
nucleic acid fragment of known sequence complimentary to the sequence to be detected; Often labeled
What is the target in hybridization assays?
complex mixture of nucleic acid fragments being tested
What is Hybridization Stringency?
degree to which non complementary sequences are tolerated during hybridization
The higher the stringency, the fewer what?
mismatches will be tolerated
What applications would require high stringency?
detecting a point mutation; need a perfect match
What applications require low stringency?
identifying multiple genes in a gene family; mismatches are tolerated
What 3 hybridization conditions determine stringency?
temperature, salt concentration, denaturing agents
Stringency increases with what?
high concentration of denaturing agents
low salt concentration
What method would we use if we had a DNA probe and a DNA target?
Southern Blot - discriminate fragments based on size and sequence
What method would we use if we had a DNA probe and a RNA target?
Northern Blot - discriminate between mRNA transcripts on the basis of sequence and size
What method would we use if we had a RNA probe and a RNA target?
in-situ hybridization (ISH) - localization of expression of specific mRNA at the tissue and cellular level
What is a a Western Blot used for?
detection of protein with antibody after electrophoresis and electro-transfer
In southern blotting, when detecting DNA bands, what does the intensity of a band reflect?
the amount of target DNA present
Would we expect to see differences between tissues in southern blots from a single patient?
No - since genomic DNA is used in southern blotting, and all a person's cells carry genomic DNA, we wouldn't expect to see differences
Can we use southern blots to discriminate between alleles carried by an individual?
Yes; if a mutation/polymorphism changes the size of the DNA fragments.
What is a Restriction Fragment Length Polymorphism (RFLP)?
a polymorphism that results in a change in the size of a restriction fragment (number of base pairs between 2 restriction sites)
Besides a single nucleotide polymorphism, what else can cause restriction fragment sizes to change?
insertion/deletion between sites
This is how other RFLPs are created
The patient with hemophilia presented with what abnormality in the periosteum of his skull?
cephalohematoma (possibly a giveaway for questions)
In general, what is the mode of inheritance for Hemophilia?
What are the 2 types of Hemophilia?
Where is the defect in Hemophilia A?
in Factor VIII (F8 gene)
Where is the defect in Hemophilia B?
in Factor IX (F9 gene)
What is hemophilia?
coagulation disorder associated with prolonged bleeding times, hemorrhage into joints and muscles
40% of severe Hemo A cases are caused by what?
an inversion due to homologous recombination between repetitive elements proximal to the F8 gene
What affect does the specific inversion have on the F8 gene in Hemo A cases?
it changes the distances between restriction sites, changing the sizes of fragments observed on Southern Blot
Would a standard PCR test detect the inversions seen in Hemo A cases?
No because all the exons remain present
In the milder form of Hemo A, how is protein function affected?
we still see residual protein function
In the more severe form of Hemo A, how is protein function affected?
we see little or no protein function
How is Hemo A treated?
by transfusion of purified/recombinant F8