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MCB1 Block 4 > Molecular Diagnostics 2 > Flashcards

Molecular Diagnostics 2 Flashcards

1
Q

What does ASO stand for?

A

Allele-Specific Oligonucleotide Hybridization

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2
Q

What is the point of ASO hybridization?

A

to detect small, sequence variants. Usually single base, but can be very small like a three base mutation. Normally point mutations

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3
Q

When doing a ASO hybridization you have to know what about mutation

A

where it is

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4
Q

ASO hybridization can be useful for screening for ____ mutations

A

frequent

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5
Q

for ASO you need how many probes are needed?

A

one for wild type

one for mutation

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6
Q

What do you do first to amplify the DNA for ASO

A

PCR amplify the product

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7
Q

Is there separation on basis of size for ASO?

A

No

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8
Q

What are you looking at in ASO hybridization, if not size

A

if there is a mutation there or not

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9
Q

What is a dot blot?

A

How you see if mutation is there in ASO hybridization

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10
Q

dark blot on normal probe tells you want

A

pt has the normal alelle

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11
Q

Why are short probes used for ASO hybridization

A

for specificity

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12
Q

Sickle cell disease is caused by what kind of muatation

A

single point mutation, A to T

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13
Q

E6V means what

A

Glutamate to valine

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14
Q

What is the specific mutation for sickle cell disease

A

E6V

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15
Q

How many probs d you need to ASO to detect sickle cell disease

A

2 - one for normal one for mutatatn

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16
Q

The probes for ASO are very small and also have what characteristic

A

Very specific - so the mutant probe will only bind to mutant and normal will only bind to normal

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17
Q

What does PCR stand for

A

polymerase chain reaction

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18
Q

What does PCR do at its basic level

A

exponentially amplify DNA

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19
Q

PCR is a _____ reaction that doubles the amount of DNA

A

cyclical

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20
Q

PCR uses what kind of polymerase

A

DNA polymerase

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21
Q

What are 2 of the negative sides of PCR?

A

need at least some sequence info to make primers

susceptible to contaminiation due to high sensitivity

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22
Q

What polymerase is most often used for PCR?

A

taq polymerase

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23
Q

PCR does DNA synthesis by a thermostable:

A

DNA polymerase

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24
Q

What is one downside of taq?

A

no proofreading

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25
Q

besides taq what DNA polymerase is often used that has proofreading

A

Pfu

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26
Q

Which is more accurate, Pfu or Taq?

A

Pfu

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27
Q

How much DNA is made each cyce of PCR

A

doubling of DNA

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28
Q

What are the steps of PCR for one cycle

A
  1. Heat to denature → single strand
  2. Cool - Primers anneal to 3’ end of one strand & to 5’ end of another strand
  3. Heat to 72 C and allow polymerization → double stranded DNA
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29
Q

Which molecules are expoentially amplified?

A

only the target sequence, the products with the extra “grey” bits at end do not get exponentially amplified

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30
Q

What is the temperature to denature in PCR

A

95 C

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31
Q

What is the temp to anneal primers in PCR

A

55-60 C

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32
Q

What are the components of PCR

A 
template DNA
2 primers
all 4 dNTPs
Buffer, Mg 2+
thermostable DNA polymerase (Taq)
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33
Q

Any reaction that uses ATP you need what ion

A

magnesium

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34
Q

What is the temp to allow polymeriztion

A

72 C

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35
Q

What is a thermocycler

A

machine that does the PCR heating and cooling process, computer controlled

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36
Q

What is the normal thermocycing sequence

A 
#1: 95 C
#2: 55 C
#3: 72 C
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37
Q

Is PCR versatile

A

yes

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38
Q

Can PCR be used for RFLP

A

yes

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39
Q

How is PCR used in sequencing

A

used first to amplify DNA before sequencing

40
Q

What is the upper limit of fragment amplification of PCR

A

1kb (pretty small)

41
Q

can you analyze large DNA regions with PCR

A

No, limited to 1kb max

42
Q

How many primers are needed in PCR

A

2

43
Q

Each primer primes what in PCR

A

synthesis of a different strand, across region of interest

44
Q

synthesis is always what direction in PCR

A

synthesis is always 5’ to 3’

45
Q

what direction are templates read in PCR

A

3’ to 5’

46
Q

What are the names of the two primers in PCR that ROdda uses

A

foward primer

reverse primer

47
Q

how long does primer need to be

A

about 20 nucleotides long

48
Q

how do we know what primer is going to be for pcr?

A

have to know the sequence

49
Q

the forward primer is what

A

identical to 5’ end of region to be amplified of the given strand

50
Q

reverse primer is what

A

complementary to the 3’ region of interested of given strand

51
Q

Draw out how primers look on PCR, where they are on 3’ and 5’

A

pg 40 in ppt

52
Q

What are 5 major applications of PCR?

A 
DNA sequencing
genotyping
diagnostics
forensics
quantifying gene expression
53
Q

How can you use PCR for genotyping

A

identifying mutations or polymorphisms in indiviuals or families

54
Q

how can you use PCR for diagnostics

A

detection of viral infections

55
Q

how can you use PCR for forensics

A

amplificatino of DNA for DNA fingerprinting

56
Q

how can you use PCR to quantify gene expression

A

measure mRNA levels (need reverse transcriptase)

57
Q

Preparation fo genomic and cDNA clones can be done via what

A

PCR

58
Q

What is RT-PCR

A

reverse transcriptase PCR

59
Q

What is RT-PCR used for?

A 
RNA virus (like HIV)
amplify mRNA of expressed genes (amplifies a cDNA)
60
Q

what is qPCR

A

Real Time/Quantitative PCR

61
Q

What is the function of qPCR?

A

it measures how much DNA is being produced in real time, it has lasers that can detect how much DNA is there after each cycle (DNA is fluorescently labeled)

62
Q

What can be used to measure microsatellites

A

PCR

63
Q

What are microsatellites

A

dynamic mutations

64
Q

How does PCR measure microsatellites

A

primers go outside of area of amplification, how big the product is that is amplified shows how big the repeat is

65
Q

Symptoms: painful swelling of fingers, splenomegaly, low hemoglobin, high reticulocytes, hemolytic anemia, blood film showing sickled erythrocytes is what disease

A

sickle cell disease

66
Q

low hemoglobin and high reticulocytes are consistent with what

A

hemolytic anemia

67
Q

what is hemolytic anemia caused by

A

early death of RBC

68
Q

what ethniciites are most affected by sickle cell?

A

african american, hispanic (but more rare)

69
Q

What mutation causes sickle cell disease?

A

E6V

70
Q

What specific mutation causes sickle cell disease

A

A→T in the HBB encoding beta globin

missense

71
Q

What codon is mutated in sickle cell disease

A

codon 6

72
Q

what is edema

A

swelling in hands and feet

73
Q

HbS is what

A

abnormal hemoglobin

74
Q

what is HbA

A

normal adult hemolobin

75
Q

What is the heterozygote advantage for sickle cell

A

heterozygotes with sickle cell survives better from malaria, so there are more mutant alleles where there are high incidents of malaria (like Africa and mediterranian)

76
Q

In E6V mutation, what is the difference in amino acids after mutation?

A

acidic to hydrophobic

77
Q

what is the trait of valine

A

hydrophobic

78
Q

what is the trait of glutamic acid (glutamate)

A

acidic

79
Q

what is mode of inheritance for sickle cell

A

AR

80
Q

What is sickle cell trait

A

heterozygote carriers can have symptoms

81
Q

What is pleiotropy

A

a genetic phenomenon where a single gene mutation leads to a variety of different systems or phenotypes throughout the body in seemingly unrelated systems

82
Q

how does sickle cell show pleiotropy

A

single base mutated → abnormal hemoglobin S → sickling
sickle cells destroyed in spleen → anemia → abnormal skull shape, weakness, splenomegaly
infarctions in blood vessels, clumping → ischaemia, thrombiss, infarction → gut, spleen, extereities, brain, kidney, lung, heart

83
Q

what is the class genetic example of pleiotropy?

A

marfan syndrom

84
Q

PCR can be used to do what analysis for sickle cell

A

RFLP

85
Q

The A→T mutation in sickle cell falls within what

A

restriction site -

86
Q

how can you do RFLP analysis for sickle cell

A

the mutation falls within restriction site

87
Q

If there is mutation what will Dde1 do?

A

It will not cut the restriction site

88
Q

What are the advantages of PCR over southern blotting

A

faster
less genomic DNA required
no labelled probe required
less expensive

89
Q

If there is no mutation what will Dde1 do?

A

will cut at AT location (where the mutation would be if there was one)

90
Q

What disease is HFE gene mutated in?

A

hereditary hemocrhomatosis

91
Q

Most cases of Turner syndrome have what mode of inheritance

A

mosaicsism

92
Q

What is genotype of turner

A

45X

93
Q

If a pt has turner syndrome and is mosiaic and gonads have 46XY what are they are risk for

A

cancer

94
Q

how do you see if a turner pt has 46XY?

A

check for Y specific sequences via PCR

95
Q

What effect does the mutation have on the restriction sites in the HFE locus?

A

The mutation introduces a restriction site. (pg 56)

MCB1 Block 4 (17 decks)

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