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GENE SEQUENCING Flashcards

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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
nucleotides that dimethylsulphate breaks
guanine
26
nucleotides that formic acid breaks
Guanine and Adenine
27
nucleic acid that hydrazine can break
thymine cytosine
28
nucleotide that hydrazine + salt can break
cytosine
29
this chemical methylates guanin
dimethylsulphate
30
this chemical protonates purines
formic acid
31
this chemical splits pyrimidine rings
thymine cytosine
32
this chemical splits only C rings
hydrazine + salt
33
reaction time of dimethylsulphate at 25*C
4 mns
34
reaction time of formic acid at 25*C
5 mns
35
reaction time of hydrazine at 25*C
8
36
reaction time of hydrazine + salt at 25*C
8mns
37
template used in maxam-gilber sequencing may either be
double stranded DNA or single-stranded DNA
38
Involves chain breakage at specific nucleotides
MAXAM-GILBERT SEQUENCING (chemical sequencing)
39
this type of sequencing uses chemicals to particularly know the order or the sequence of nucleic acids
MAXAM-GILBERT SEQUENCING (chemical sequencing)
40
Before the DNA template is cleaved into fragments, first, it must be radio-labelled. An example of radioactive label is the
phosphorus 32 (P32) sulfur 32
41
Chemical sequencing needs a __ in order for base modifiers to function
radio label
42
are the ones placed on the tubes
aliquots
43
are the ones written on the tube
base modifiers
44
tracking dye/loading dye sample we can use in maxam-gilber chemical sequencing
bromphenol blue xylene cyanol loading dye
45
loading dyes are used to monitor the migration of the fragments in the electrophoresis
Bromophenol blue and xylene cyanol
46
Most notable disadvantages of MAXAM-GILBERT CHEMICAL SEQUENCING
chemicals used are hazardous time consuming
47
is a strong reducing agent that cleaves your DNA template at specific sites breaking down the specific nucleotides
10% piperidine
48
after breaking the nucleotide using 10% piperidine, we must put it in the electrophoresis and we'll use ____
polyacrylamide gel 6% - 20%
49
the formed band in dna dequencing after electrophoresis is called as
sequencing ladder
50
running time of ___ 50 kilo bases
2 hrs
51
running time of ___ longer kilo bases
7-8 hrs
52
SANGER SEQUENCING is developed by
frederick sanger
53
pcr based method of dna sequencing
SANGER SEQUENCING
54
gold standard for dna sequencing
SANGER SEQUENCING
55
a two-time recipient of the Nobel Prize
Frederick Sang Frederick Sanger
56
SANGER SEQUENCING is also called as
dideoxychain termination sequencing method
57
why is sanger sequencing called as dideoxy chain termination sequencing method
because in the process, it is using DDNTP Dideoxynucleotide triphosphate (ddNTP)
58
the one used for breaking of the growing ladder of DNA for sanger sequencing is the
Dideoxynucleotide triphosphate (ddNTP)
59
4 types of dNTPs
ddATP ddTTP ddCTP ddGTP
60
ratio of the primer and template in sanger sequencing
1:1
61
what are the components of sanger sequencing per tube
per tube add 1:1 primer and template add each corresponding add the 4 normal ddNTP DNA polymerase(sequencing enzyme),
62
type of DNA we use for sanger sequencing
single stranded DNA - primer will not be able to attach to double stranded
63
give the summary of the process of sanger sequencing
denaturation attachment of primer extension by DNA pol termination gel electrophoresis to produce product
64
difference of normal dNTP to ddNTP
dNTP has oxygen on the 3rd carbon atom ddNTP has no oxygen on the 3rd carbon atom
65
explain the relation of ddNTP and phosphodiester bond
ddNTP has no oxygen on the 3rd carbon atom which means the phosphodiester bond which primary purpose is to attach the 3`end 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
if the concentration of the ddNTPs is too high, what will happen to the dna sequence
polymerization will terminate frequently and therefore you will have a short sequence read
67
if the concentration of the ddNTPs is too low, what will happen to the dna sequence
it will result in a long sequence read
68
ano gagawin if the band is too close to one another making it hard to read
decrease the use of polyacrylamide gel
69
sanger sequencing usually takes how many mns or hrs
20 mns
70
after sequencing in sanger sequencing, we can add ____
stock buffer -- 20 millimolar EDTA to chelate Mg and Calcium cations -- formamide
71
purpose of Mg in sequencing
enhance Taq polymerase
72
purpose of formamide
denatures the product of sequencing reaction from ds to ss
73
loading dye used in sanger sequencing
bromphenol and xylene cyanol
74
sanger method comparison to maxam gilbert give the characteristics of sanger sequencing
enzymatic requires dna synthesis termination of chain elongation automation single stranded DNA
75
maxam gilbert comparison to sanger method give the characteristics of maxam gilbert sequencing
chemical requires dna cleavage breaks dna at different nucleotides automation not available dsDNA or ssDNA
76
DNA sequencing has been applied in ___ science to identify particular individual because every individual has unique sequence of his/her DNA
forensics
77
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.
forensic application
78
how sequencing applies to agriculture
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
how can sequencing be useful in medicine
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
2 types of Automated Sanger sequencing
cycle sequencing fluorescent dye sequencing
81
difference of cycle sequencing to manual sanger sequencing
same process but is conducted on a thermal cycler
82
are multicyclic molecules that absorb and emit fluorescent light a specific wavelengths
fluorescent dye
83
example of fluorescent dye
fluorescein rhodamine bodipy dye (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)
84
2approaches of fluorescent sequencing
dying the primer dying the terminator NTPs or ddNTPs
85
color of adenine
green
86
color of cytosine
blue
87
color of thymine
red
88
color of guanine
yellow or black
89
between the 4 tubes (dying the primer/dying the terminator) which one will you choose
dying the terminator
90
is the automated sanger sequencing of choice
dying the terminator
91
excites the fluorescent dye, activating the fluorescent dye to emit a visible dye
laser
92
instead of using different laid gel we can use ___ in a single method
capillary gel electrophoresis
93
is based on the generation of light signal through release of pyrophosphate (PPi) on the nucleotide addition
Pyrosequencing
94
DNA + dNTP will yield
pyrophosphate (PPi) DNAn+1 + PPi
95
the pyrophosphate (PPi) cleaved from dNTP and DNA is used to generate __
generate ATP from adenosine phosphosulfate (APS)
96
the ATP that is generated by the PPi from adenosine phosphosulfate will then be use to ___
together with luciferase, ATP will generate light by conversion of LUCIFERIN to OXYLUCIFERIN
97
enzyme that will catalyze the conversion of luciferin to oxyluciferin
luciferase
98
enzyme that will catalyze the production of ATP from adenosine phosphosulfate and pyrosulfate
adenosine sulfurylase
99
3 steps of pyrosequencing
o Polymerase o Sulfurylase o Luciferase
100
apyrase came from what insect
dragonfly
101
this enzyme that is important in resequencing as it can degrade or remove dNTP that are misincorporated
apyrase
102
detector of pyrosequencing
luminometer
103
use of pyrosequencing
for short to moderate sequence analysis
104
application of pyrosequencing
mutation detection infectious disease detection dna methylation analysis
105
used to detect cytosine methylation in DNA
bisulfite
106
Important in epigenetics
bisulfite
107
it deaminates cytosine, making uracil
bisulfite
108
Methylated cytosine is NOT CHANGED by bisulfite treatment true or false
true
109
a normal cytosine after bisulfite treatment will result to ___
convertion to uracil
110
application of bisulfite sequencing
regulation of gene expression chromatin structure detection of cell differentiation
111

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