L10 Molecular Diagnosis Flashcards
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what are the 2 main uses of Linkage analysis?
- when the mutation is not known
- when the disease exhibits allelic heterogeneity
for linkage analysis, the gene of interest has to be linked to a _______
known marker locus (polymorphic marker)
= performed by recombination mapping
define crossover
during prophase I of meiosis, homologous chromosomes line up and exchange portions of DNA - it looks like the chromosomes are kissing!
Loci that are far apart experience a crossover _____ frequently
more frequently – like A and C from lecture example
also, high recombination frequency
loci that are close to each other experience a crossover ____ frequently
less frequently – like A and B from lecture example
also, low recombination frequency
why is linkage analysis being less important?
because sequencing data is accumulating
what does linkage analysis require?
- large family with several affected individuals
- linkage map (markers near the region of interest)
linkage analysis may then be used to identify a family member who has a high probability of inheriting the causative mutation by determining who carries a _______ which is usually inherited with the disease causing mutation on the chromosome
polymorphic marker
if 2 loci are close together on a chromosome, what are the chances of recombination?
low = Linked loci
a marker locus close to the _____ has to be identified
mutant gene
initially, the marker is identified in a family and later the marker may sometimes be applied for use in the population under what condition?
the marker must be closely linked to the mutant gene
how is linkage analysis carried out
- marker is mapped in all family members = affected and not affected
- marker is analyzed in all the affected persons in the family
- marker may be linked to disease is all affected members have marker
- marker should be absent in all unaffected members
how is identification of the actual disease causing gene accomplished?
sequencing nearby regions of the genome (in the past), or by use of the human genome databases (current)
_____status may be determined by linkage analysis when presented with an autosomal recessive disease
carrier status
how can you determine carrier status from linkage analysis in autosomal recessive disorders?
obtain DNA samples and analyze by linkage to RFLP (restriction fragment length polymorphism) marker
if the marker is linked, identify the markers linkage - what allele A or a is being passed down and causing the disease?!
_____ may result in a diseased individual without the marker or result in a non-affected individual with the disease marker
recombination
the farther the marker is form the disease locus, the chances of recombination ___
increases
what are the two big challenges of using linkage analysis?
- the occurrence of a recombination event during meiosis = recombination event
- same phenotype but different loci (in a different family) = locus heterogeneity
if a mutation interferes with a restriction site, the RFLP allows____
direct detection
the DNA of 2 unrelated individuals will have a single bp different every ____ bp
1000 bp
1 polymorphism : 1000 bp
there are about 3,000,000 differences = 99.9% similar….
what effects can base pair differences between individuals have
- sometimes effects a restriction site
- maybe creation of restriction site
- maybe loss of restriction site
digestion of DNA from different individuals may result in ____
different patterns of DNA fragments
what is a restriction site - wikipedia
Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific (4-8 base pairs in length)[1] sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences[2] (because restriction enzymes usually bind as homodimers), and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby.
what is the basis of RFLP (2)
- restriction site is recognized by a restriction endonuclease - can be palindrome
- NOT a PALINDROME and not recognized as a restriction site by a restriction endonuclease
what technique was used to originally obtain RFLP?
southern blot
what are the disadvantages of using southern blot?
- procedure is long - takes 24 hours!
- may not be automated - humans have to do it
- uses radioactivity
- expensive
rarely used these days!
probe position may affect what?
banding patterns
RFLP data may be generated by what
PCR
PCR + cut with restriction enzyme + separate by agarose gel electrophoresis
what is a direct test for RFLP
when the mutation is at the actual site for the restriction enzymes = sickle cell anemia
sickle allele is interesting in that the mutation causes ____ restriction site but _____
1
destroys
Describe ASO (Allele specific oligoneucleotide) probes
short oligonucleotide sequences (~15 bp long) that specifically bind to a single allele of a gene
when is ASO probes possible?
only if the exact mutation in the gene has been identified
why are ASO tests becoming less useful?
disorders are exhibiting increase allelic or genetic heterogeneity
you need to know what your looking for in order to design the test…
what disorders are ASO test useful for?
CF and hemochromatosis + sickle cell anemia
what is the most common mutation (3 bp deletion)in CF
delta F508 = single codon deletion
70% of carriers harbor this allele
50% of affected individuals are homozygous for this allele
what is PCR used to detect?
single nucleotide changes
how does PCR detect single nucleotide changes
by designing primers which hybridize to the region containing the polymorphism
what is the function of internal controls in PCR
they ensure that the PCR is working properly
