L10 Molecular Diagnosis

Question 1

what are the 2 main uses of Linkage analysis?

Answer 1

• when the mutation is not known

- when the disease exhibits allelic heterogeneity

Question 2

for linkage analysis, the gene of interest has to be linked to a _______

Answer 2

known marker locus (polymorphic marker)

= performed by recombination mapping

Question 3

define crossover

Answer 3

during prophase I of meiosis, homologous chromosomes line up and exchange portions of DNA - it looks like the chromosomes are kissing!

Question 4

Loci that are far apart experience a crossover _____ frequently

Answer 4

more frequently – like A and C from lecture example

also, high recombination frequency

Question 5

loci that are close to each other experience a crossover ____ frequently

Answer 5

less frequently – like A and B from lecture example

also, low recombination frequency

Question 6

why is linkage analysis being less important?

Answer 6

because sequencing data is accumulating

Question 7

what does linkage analysis require?

Answer 7

• large family with several affected individuals

- linkage map (markers near the region of interest)

Question 8

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

Answer 8

polymorphic marker

Question 9

if 2 loci are close together on a chromosome, what are the chances of recombination?

Answer 9

low = Linked loci

Question 10

a marker locus close to the _____ has to be identified

Answer 10

mutant gene

Question 11

initially, the marker is identified in a family and later the marker may sometimes be applied for use in the population under what condition?

Answer 11

the marker must be closely linked to the mutant gene

Question 12

how is linkage analysis carried out

Answer 12

• 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

Question 13

how is identification of the actual disease causing gene accomplished?

Answer 13

sequencing nearby regions of the genome (in the past), or by use of the human genome databases (current)

Question 14

_____status may be determined by linkage analysis when presented with an autosomal recessive disease

Answer 14

carrier status

Question 15

how can you determine carrier status from linkage analysis in autosomal recessive disorders?

Answer 15

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?!

Question 16

_____ may result in a diseased individual without the marker or result in a non-affected individual with the disease marker

Answer 16

recombination

Question 17

the farther the marker is form the disease locus, the chances of recombination ___

Answer 17

increases

Question 18

what are the two big challenges of using linkage analysis?

Answer 18

• the occurrence of a recombination event during meiosis = recombination event
• same phenotype but different loci (in a different family) = locus heterogeneity

Question 19

if a mutation interferes with a restriction site, the RFLP allows____

Answer 19

direct detection

Question 20

the DNA of 2 unrelated individuals will have a single bp different every ____ bp

Answer 20

1000 bp

1 polymorphism : 1000 bp
there are about 3,000,000 differences = 99.9% similar….

Question 21

what effects can base pair differences between individuals have

Answer 21

• sometimes effects a restriction site
• maybe creation of restriction site
• maybe loss of restriction site

Question 22

digestion of DNA from different individuals may result in ____

Answer 22

different patterns of DNA fragments

Question 23

what is a restriction site - wikipedia

Answer 23

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.

Question 24

what is the basis of RFLP (2)

Answer 24

• 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

Question 25

what technique was used to originally obtain RFLP?

Answer 25

southern blot

Question 26

what are the disadvantages of using southern blot?

Answer 26

• procedure is long - takes 24 hours!
• may not be automated - humans have to do it
• uses radioactivity
• expensive

rarely used these days!

Question 27

probe position may affect what?

Answer 27

banding patterns

Question 28

RFLP data may be generated by what

Answer 28

PCR

PCR + cut with restriction enzyme + separate by agarose gel electrophoresis

Question 29

what is a direct test for RFLP

Answer 29

when the mutation is at the actual site for the restriction enzymes = sickle cell anemia

Question 30

sickle allele is interesting in that the mutation causes ____ restriction site but _____

Answer 30

1

destroys

Question 31

Describe ASO (Allele specific oligoneucleotide) probes

Answer 31

short oligonucleotide sequences (~15 bp long) that specifically bind to a single allele of a gene

Question 32

when is ASO probes possible?

Answer 32

only if the exact mutation in the gene has been identified

Question 33

why are ASO tests becoming less useful?

Answer 33

disorders are exhibiting increase allelic or genetic heterogeneity

you need to know what your looking for in order to design the test…

Question 34

what disorders are ASO test useful for?

Answer 34

CF and hemochromatosis + sickle cell anemia

Question 35

what is the most common mutation (3 bp deletion)in CF

Answer 35

delta F508 = single codon deletion
70% of carriers harbor this allele
50% of affected individuals are homozygous for this allele

Question 36

what is PCR used to detect?

Answer 36

single nucleotide changes

Question 37

how does PCR detect single nucleotide changes

Answer 37

by designing primers which hybridize to the region containing the polymorphism

Question 38

what is the function of internal controls in PCR

Answer 38

they ensure that the PCR is working properly