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MCB1 Block 4 > Molecular Diagnositcs 3 > Flashcards

Molecular Diagnositcs 3 Flashcards

1
Q

What is an alternative approach to ASO hybridization

A

allele-specific PCR

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2
Q

What is MLPA

A

combining lots of PCRs together

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3
Q

What is the principle in allele-specific PCR

A

PCR primers

looking for allele-specific mutations (like ASO but primers )

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4
Q

If you use normal primer on mutatnt allele what kind of product?

A

no PCR product

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5
Q

If you use mutant primer on normal allele what kind of product?

A

no PCR product

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6
Q

If there’s a mismatch at the 3’ end what kind of product

A

no PCR product

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7
Q

What kind of primer do you use for reverse primer for allele-specific pCR

A

allele-specific reverse primer

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8
Q

What kind of product do you look at to interpret Allele-specific PCR

A

bands instead of drops

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9
Q

Can you use reverse or forward primer for allele-specific PCR?

A

you can use either, have to choose which one you want to be

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10
Q

What is multiplex PCR

A

method to scan commonly deleted exons. It can scan large regions of genome
It does lots of PCR reactions combined into one test tube, so multiple done at once

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11
Q

Why do we use multiplex PCR? Give an example

A

big deletions, like duchenne muscular dystrophy. DMD is too big to sequence normal way.

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12
Q

How many primers are used for multiplex PCR

A

multiple primers are used, you are doing a ton of reactions at once. You don’t know how big deletion is so you use a bunch of primers to get an idea of how big deletion is

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13
Q

What kind of method could be used to diagnose DMD?

A

multiplex PCR

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14
Q

What does MLPA stand for

A

multiplex ligation-dependent probe amplification

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15
Q

What method can be used to identify carrier mothers for DMD

A

MLPA

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16
Q

In typical MLPA how many primers can be used

A

up to 40

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17
Q

Instead of a single probe what do you use for MLPA

A

two primers that hybridize end to end

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18
Q

In MLPA when two primers hybridize what do they form in the middle

A

nic

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19
Q

How do you combine the nic formed from two primers hybridizing in MLPA

A

ligase

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20
Q

ligase only works if what (in MLPA)

A

template DNA is there

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21
Q

The amount of ligated product will be directly proportional to what in MLPA

A

amount of template used

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22
Q

The primers all have the same what in MLPA

A

PCR binding site

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23
Q

How do you distinguish b/w the different primers in MLPA?

A

stuffer sequence

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24
Q

What is stuffer seuence?

A

little pieces of DNA that changes the size

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25
Q

How do you distinguish the different products in MLPA

A

they all have a different size, can run a gel and they are all different size

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26
Q

Are primers in MLPA flerourescently labeled?

A

yes

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27
Q

Each primer set represents a different what in MLPA

A

exon

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28
Q

What’s the biggest difference b/w MLPA and multiplex pCR

A

MLPA can scan bigger region
can determine more clearly if pt is heterozygous carrier
MLPA is more quantitative

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29
Q

What are the advantages of MLPA

A

scanning large regions (can use 40 primers at a time)

more quantitative

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30
Q

What is the negative part of MLPA

A

only detects copy number, doesn’t detect chromosomal rearrangmeents

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31
Q

What does FISH stand for

A

fluorescent In-situ hybridization

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32
Q

How do you determine what approach to use for detecting deletion?

A

depends on how big deletion is

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33
Q

What do you use to detect small deletions

A

ASO or PCR

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34
Q

What do you use to detect medium deletions (100s of BP)

A

PCR, Southern blotting, MLPA, CGH

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35
Q

What do you use to detect large deletions (10’s of kb)

A

Southern blotting, MLPA, CGH

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36
Q

What do you use to detect VERY large deletions (500 kb - 3 mb)

A

FISH, CGH

37
Q

What does In-situ mean

A

looking in a cell or tissues

38
Q

What is the target in FISH

A

cellular chromosomal DNA

39
Q

what is the probe in FISH

A

BAC

40
Q

What does BAC stand for

A

bacterial artifical chromosomes

41
Q

what is size of BAC

A

100-200 kb

42
Q

What is BAC how is it a probe

A

vector used to carry large amounts of DNA

43
Q

How can you visualize in FISH

A

fluorescent microscopy

44
Q

If probe is larger than deletion, what will happen in FISH

A

it won’t discriminate - it will still hybridize to the one you don’t want it to, if’s so big it doesn’t matter that there is deletion

45
Q

What is metaphse FISH

A

when cells are growing in culture synchonize and arrest them in metaphase

46
Q

what is the advantage of metaphase FISH

A

can see chromosomes - can see location and if chromosome is there

47
Q

what is interphase fish

A

don’t arrest cells

48
Q

what is the advantage of interphase fish

A

don’t have to arrest them

49
Q

what is the disadvantage of interphase fish

A

can’t see chromosomes or location

50
Q

What is chromosome painting

A

FISH probes span the entire length of choromsome

51
Q

In chromosome painting what is labeled

A

the entire chromsome

52
Q

what is chromosmal painting good for detecting

A

specific, known chromosomal rearrangements

53
Q

What does SKY stand for

A

spectral karyotyping

54
Q

What is SKY

A

all chromsomal are painted different colors

55
Q

what is the function of SKY

A

useful for identifying unknown chromosomal rearrangements

56
Q

What does VSD stand for

A

ventricular septal defect

57
Q

22q11 How would you use FISH

A

two different probes
a control probe
test probe specific for the region we are looking for

58
Q

What does metaphase FISH test for

A

If part of chromosome is there or not

59
Q

What is the other name of 22q11 Deletion syndrome

A

DiGeorge Syndrome

60
Q

What is the mode of inheritance for DiGeorge?

A

AD

61
Q

CATCH22 is mneominic for symptoms for DiGeorge, liste them

A 
cardiac defects
abnormal facies
thymic hypoplasia
cleft palate
hypocalemia frmo parathyroid aplasia
chromosome 22
62
Q

What test can you use to confirm Down Syndrome?

A

FISH - can use interphase FISh

63
Q

What probes would you use to test for Down Syndrome using FISH

A

one labeling chromosome 18

One labeling chromosome 21

64
Q

What does CGH stand for

A

comparative genome hybridization

65
Q

What can CGH be used for

A

looking for changes in copy number of specific sites

can scan entire genome

66
Q

What are the limitations of CGH

A

doesn’t detect balanced rearrangeement, can only detect copy number. Can only detect deletions and insertions

67
Q

Describe process of doing array-CGH

A

label test and control two different colors and hybridize them against microarray. if they both bind to a spot and one is red and one is green then it will be yellow

68
Q

In array-CGH the ratio of red and green determine what

A

if pts DNA is over or udner represented

69
Q

If there is a lot of green (green is control) what does that mean array-CGH

A

control DNA is present, test DNA is not, so there is deletion

70
Q

If there is red (red is test) in array-CGH what does that mean?

A

duplication

71
Q

If there is yellow in array-CGH what does that mean

A

no difference, same amount of test and control DNA

72
Q

If spots go lower on CGH output what does it mean

A

deletion

73
Q

If spots go higher on CGH output what does that mean

A

duplication

74
Q

How do you find out where chromosomal abnormality is if you do not know

A

CGH

75
Q

Spots going up on chromosome 12 means what

A

duplication at the location that the dots go up

76
Q

What is DNA sequencing

A

the final solution for genetic testing.

can look at highest level and see where mutation is

77
Q

What are the limitations to DNA sequencing

A

can only look at a specific area at a time

expensive

78
Q

When would you use DNA sequencing

A

AD conditions
ex: marfan syndrome, osteogensis imperfecta
AR where the mutation is not common, where it’s highly variable b/w families

79
Q

What is the basis of dideoxy sequencing

A

uses dideoxynucleotide - it stops where they are

80
Q

what is the difference in dideoxynucleotide

A

doesnt have 2 or 3’ OH

81
Q

in electrophoretic separation and you read gel from bottom to top, what diretion in sequence are you reading

A

5’ to 3’

82
Q

The sequence of the template strand is what in comparison to the gel

A

complementary

83
Q

When you read a gel what sequence are you reading

A

NOT the template strand, have to take complement of the gel you’re reading

84
Q

What is dye-termination sequencing

A

the theory is the same as dideoxynucleotide, but don’t have to use four different reqctions, combined in a single reaction

85
Q

How do you distinguish b/w different fragments in dye-termination sequencing?

A

can tell termination by color

86
Q

What do you run DNA through in dye-termination sequencing

A

run through capillary electrophoresis coupled to a fluorescence detector

87
Q

What do you run DNA through in dideoxy sequencing

A

acrilymide gel

88
Q

What does a peak mean in dye-termination sequencing

A

peaks of specific colors correspond to terminations at specific bases

89
Q

How do you read peak graph from dye-termination sequencing

A

read left to right 5’ to 3’

MCB1 Block 4 (17 decks)

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