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MODX > GENE SEQUENCING > Flashcards

GENE SEQUENCING Flashcards

1
Q

is a process of knowing the correct order
of nucleotides in a particular segment of DNA

A

DNA sequencing

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2
Q

B/L/A/S/T means

A

basic local alignment search tool

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3
Q

the order of nucleotides in
the DNA molecule is used in the medical laboratory for a
variety of purposes such as

A

detecting mutations
typing microorganism
identifying human haplotypes
designating polymorphisms

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4
Q

changes in the exact sequences of our nucleic acid with regards to
greater than 1% of population.

A

Polymorphisms

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5
Q

changes in the sequence of our
nucleic acid with respect to less than 1% of our population.

A

mutation

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6
Q

DNA sequencing is can be used in targeted strategies called as

A

targeted therapies

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7
Q

is the process of determining the
precise order of nucleotides within a DNA molecule

A

DNA Sequencing

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8
Q

DNA Sequence

is just a part or portion of the genome of
a particular organism or sample.

true or false

A

true

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9
Q

is the most definitive molecular method to
identify genetic lesions or mutations and
polymorphisms

A

Direct determination of the nucleotide sequence, or DNA sequencing

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10
Q

2 types of manual sequencing

A

maxam-gilbert sequencing
sanger sequencing

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11
Q

Two types of sequencing methods were concurrently
developed in the what year

o Maxam–Gilbert sequencing
o Sangersequencing

A

1970s

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12
Q

a type of sequencing that is conducted in viruses

A

whole genome sequencing

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13
Q

Automated sequencing is based on

A

Sanger method (dideoxy chain)

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14
Q

is used in whole genome sequencing and
in sequencing several samples of DNA
simultaneously.

A

next generation sequencing

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15
Q

a type of sequencing that uses several platforms or approaches depending on the manufacturer of the machine

A

NGS or next generation sequencing

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16
Q

Next Generation Sequencing is also known as

A

massive parallel sequencing

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17
Q

Human genome project started in the year of __ and ended in the year of ___.

A

1990; 2003

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18
Q

By th etime of 2003, only ___% of human genome sequence is studied

A

80

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19
Q

In the year of __, 100% of DNA sequencing
has been studied (32 years to complete the study of human genome)

A

2022

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20
Q

how many years does the study of human genome is completed

A

32 yrs

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21
Q

MAXAM-GILBERT SEQUENCING is developed by whom

A

allam maxam
walter gilbert

1976-1977

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22
Q

was the first widely adopted
method for DNA sequencing

A

Maxam–Gilbert sequencing

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23
Q

Method based on chemical modification of DNA and
subsequent cleavage at specific nitrogenous bases

A

maxam-gilber sequencing

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24
Q

4 chemicals we can use in DNA sequencing

A

dimethylsulphate
formic acid
hydrazine
hydrazine + salt

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25
Q

nucleotides that dimethylsulphate breaks

A

guanine

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26
Q

nucleotides that formic acid breaks

A

Guanine and Adenine

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27
Q

nucleic acid that hydrazine can break

A

thymine
cytosine

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28
Q

nucleotide that hydrazine + salt can break

A

cytosine

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29
Q

this chemical methylates guanin

A

dimethylsulphate

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30
Q

this chemical protonates purines

A

formic acid

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31
Q

this chemical splits pyrimidine rings

A

thymine
cytosine

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32
Q

this chemical splits only C rings

A

hydrazine + salt

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33
Q

reaction time of dimethylsulphate at 25*C

A

4 mns

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34
Q

reaction time of formic acid at 25*C

A

5 mns

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35
Q

reaction time of hydrazine at 25*C

A

8

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36
Q

reaction time of hydrazine + salt at 25*C

A

8mns

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37
Q

template used in maxam-gilber sequencing may either be

A

double stranded DNA or single-stranded DNA

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38
Q

Involves chain breakage at specific nucleotides

A

MAXAM-GILBERT SEQUENCING
(chemical sequencing)

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39
Q

this type of sequencing uses chemicals to particularly know the order or
the sequence of nucleic acids

A

MAXAM-GILBERT SEQUENCING
(chemical sequencing)

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40
Q

Before the DNA template is cleaved into fragments, first, it must be radio-labelled. An example of radioactive label is the

A

phosphorus 32 (P32)
sulfur 32

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41
Q

Chemical sequencing needs a __ in order for base modifiers to function

A

radio label

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42
Q

are the ones placed on the tubes

A

aliquots

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43
Q

are the ones written on the tube

A

base modifiers

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44
Q

tracking dye/loading dye sample we can use in maxam-gilber chemical sequencing

A

bromphenol blue
xylene cyanol loading dye

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45
Q

loading dyes are
used to monitor the migration of the fragments in the
electrophoresis

A

Bromophenol blue and xylene cyanol

46
Q

Most notable disadvantages of MAXAM-GILBERT CHEMICAL SEQUENCING

A

chemicals used are hazardous
time consuming

47
Q

is a strong reducing agent that cleaves
your DNA template at specific sites breaking down the
specific nucleotides

A

10% piperidine

48
Q

after breaking the nucleotide using 10% piperidine, we must put it in the electrophoresis and we’ll use ____

A

polyacrylamide gel 6% - 20%

49
Q

the formed band in dna dequencing after electrophoresis is called as

A

sequencing ladder

50
Q

running time of ___ 50 kilo bases

A

2 hrs

51
Q

running time of ___ longer kilo bases

A

7-8 hrs

52
Q

SANGER SEQUENCING is developed by

A

frederick sanger

53
Q

pcr based method of dna sequencing

A

SANGER SEQUENCING

54
Q

gold standard for dna sequencing

A

SANGER SEQUENCING

55
Q

a two-time recipient of
the Nobel Prize

A

Frederick Sang Frederick Sanger

56
Q

SANGER SEQUENCING is also called as

A

dideoxychain termination sequencing method

57
Q

why is sanger sequencing called as dideoxy chain termination sequencing method

A

because in the process, it is using DDNTP

Dideoxynucleotide triphosphate (ddNTP)

58
Q

the one used for breaking of the growing ladder of DNA for sanger sequencing is the

A

Dideoxynucleotide triphosphate (ddNTP)

59
Q

4 types of dNTPs

A

ddATP
ddTTP
ddCTP
ddGTP

60
Q

ratio of the primer and template in sanger sequencing

A

1:1

61
Q

what are the components of sanger sequencing per tube

A

per tube add 1:1 primer and template
add each corresponding
add the 4 normal ddNTP
DNA polymerase(sequencing enzyme),

62
Q

type of DNA we use for sanger sequencing

A

single stranded DNA - primer will not be able to attach to double stranded

63
Q

give the summary of the process of sanger sequencing

A

denaturation
attachment of primer
extension by DNA pol
termination
gel electrophoresis to produce product

64
Q

difference of normal dNTP to ddNTP

A

dNTP has oxygen on the 3rd carbon atom
ddNTP has no oxygen on the 3rd carbon atom

65
Q

explain the relation of ddNTP and phosphodiester bond

A

ddNTP has no oxygen on the 3rd carbon atom which means the phosphodiester bond which primary purpose is to attach the 3end to the 5 end of other nucleotide will be terminated.

no oxygen, no phosphodiester bond. That makes ddNTP’s primary function is to break the nucleotide or terminate the DNA ladder chain

66
Q

if the concentration of the ddNTPs is too high, what will happen to the dna sequence

A

polymerization will terminate frequently and therefore you will have a short sequence read

67
Q

if the concentration of the ddNTPs is too low, what will happen to the dna sequence

A

it will result in a long sequence read

68
Q

ano gagawin if the band is too close to one another making it hard to read

A

decrease the use of polyacrylamide gel

69
Q

sanger sequencing usually takes how many mns or hrs

A

20 mns

70
Q

after sequencing in sanger sequencing, we can add ____

A

stock buffer

– 20 millimolar EDTA to chelate Mg and Calcium cations
– formamide

71
Q

purpose of Mg in sequencing

A

enhance Taq polymerase

72
Q

purpose of formamide

A

denatures the product of sequencing reaction

from ds to ss

73
Q

loading dye used in sanger sequencing

A

bromphenol and xylene cyanol

74
Q

sanger method comparison to maxam gilbert
give the characteristics of sanger sequencing

A

enzymatic
requires dna synthesis
termination of chain elongation
automation
single stranded DNA

75
Q

maxam gilbert comparison to sanger method
give the characteristics of maxam gilbert sequencing

A

chemical
requires dna cleavage
breaks dna at different nucleotides
automation not available
dsDNA or ssDNA

76
Q

DNA sequencing has been applied in ___ science to identify particular individual because
every individual has unique sequence of his/her DNA

A

forensics

77
Q

t is particularly ed to identify the criminals by finding some proof from the crime scene in the form
of hair, nail, skin or blood samples.

A

forensic application

78
Q

how sequencing applies to agriculture

A

ü The mapping and sequencing of the whole genome of microorganisms has allowed the agriculturists to
make them useful for the crops and food plants.

79
Q

how can sequencing be useful in medicine

A

ü In medical research, DNA sequencing can be used to detect the genes which are associated with some heredity or acquired diseases.

ü Scientists use different techniques of genetic engineering like gene therapy to identify the defected genes and replace them with the healthy ones.

80
Q

2 types of Automated Sanger sequencing

A

cycle sequencing
fluorescent dye sequencing

81
Q

difference of cycle sequencing to manual sanger sequencing

A

same process but is conducted on a thermal cycler

82
Q

are multicyclic molecules that absorb and emit fluorescent light a
specific wavelengths

A

fluorescent dye

83
Q

example of fluorescent dye

A

fluorescein
rhodamine
bodipy dye (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)

84
Q

2approaches of fluorescent sequencing

A

dying the primer
dying the terminator NTPs or ddNTPs

85
Q

color of adenine

A

green

86
Q

color of cytosine

A

blue

87
Q

color of thymine

A

red

88
Q

color of guanine

A

yellow or black

89
Q

between the 4 tubes (dying the primer/dying the terminator) which one will you choose

A

dying the terminator

90
Q

is the automated sanger sequencing of choice

A

dying the terminator

91
Q

excites the fluorescent dye, activating the fluorescent dye to emit a visible dye

A

laser

92
Q

instead of using different laid gel we can use ___ in a single method

A

capillary gel electrophoresis

93
Q

is based on the generation of
light signal through release of pyrophosphate
(PPi) on the nucleotide addition

A

Pyrosequencing

94
Q

DNA + dNTP will yield

A

pyrophosphate (PPi)

DNAn+1 + PPi

95
Q

the pyrophosphate (PPi) cleaved from dNTP and DNA is used to generate __

A

generate ATP from adenosine phosphosulfate (APS)

96
Q

the ATP that is generated by the PPi from adenosine phosphosulfate will then be use to ___

A

together with luciferase, ATP will generate light by conversion of LUCIFERIN to OXYLUCIFERIN

97
Q

enzyme that will catalyze the conversion of luciferin to oxyluciferin

A

luciferase

98
Q

enzyme that will catalyze the production of ATP from adenosine phosphosulfate and pyrosulfate

A

adenosine sulfurylase

99
Q

3 steps of pyrosequencing

A

o Polymerase
o Sulfurylase
o Luciferase

100
Q

apyrase came from what insect

A

dragonfly

101
Q

this enzyme that is important in resequencing as it can degrade or remove dNTP that are misincorporated

A

apyrase

102
Q

detector of pyrosequencing

A

luminometer

103
Q

use of pyrosequencing

A

for short to moderate sequence analysis

104
Q

application of pyrosequencing

A

mutation detection
infectious disease detection
dna methylation analysis

105
Q

used to detect cytosine methylation in DNA

A

bisulfite

106
Q

Important in epigenetics

A

bisulfite

107
Q

it deaminates cytosine, making uracil

A

bisulfite

108
Q

Methylated cytosine is NOT CHANGED by bisulfite treatment

true or false

A

true

109
Q

a normal cytosine after bisulfite treatment will result to ___

A

convertion to uracil

110
Q

application of bisulfite sequencing

A

regulation of gene expression
chromatin structure
detection of cell differentiation

111
Q
A

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