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Molecular Biology Lec > LESSON 4 BASIC METHODS & TOOLS IN MOLECULAR BIOLOGY & DIAGNOSTICS > Flashcards

LESSON 4 BASIC METHODS & TOOLS IN MOLECULAR BIOLOGY & DIAGNOSTICS Flashcards

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1
Q

type of specimen collected when testing for current or past infection with SARS-CoV-2 is based on the

A

test being performed and its manufacturer’s instructions

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2
Q

• CDC recommendation for initial dx test:

A

upper respiratory specimen

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3
Q

– easier to do

A

• Nasopharyngeal swab and Oropharyngeal swab

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4
Q

• Specimen with the highest viral load is nasopharyngeal wash/aspirate

A

• Nasopharyngeal swab and Oropharyngeal swab

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5
Q

Work within [?] of patients

A

6ft

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6
Q

• Maintain proper

A

infection control

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7
Q

• Follow

A

standard precautions

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8
Q

• Ppe with [?] or higher level of respirator

A

n95

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9
Q

The respirator must be put on [?] and worn during the exposure.

A

correctly

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10
Q

The respirator filter must capture more than [?] of the particles from the air that passes through it.

A

95%

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11
Q

The respirator must [?] against the user’s face to ensure that there are no gaps between the user’s skin and respirator seal.

A

fit snugly

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12
Q

is a critical component to a respiratory protection program.

A

Fit testing

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13
Q

OSHA requires an initial respirator fit test to identify the right [?] respirator for each worker.

A

model, style, and size

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14
Q

ensure that users continue to receive the expected level of protection.

A

• Annual fit tests

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15
Q

Collect as soon as possible after symptoms begin (ideally within?)

A

7 days

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16
Q

PCR - can detect virus from day [?] (virus is already shedding and multiplying; there are already remnants of RNA)

A

1 to day 7

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17
Q
  • Antibody production against SARs-Cov 2 genes takes[?]
A

17 days/2 weeks

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18
Q
  • Antigen can be detected during the
A

first 4 days

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19
Q

Ideally before [?] are administered

A

V antiviral medications

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20
Q

Collect multiple specimens on

A

multiple days

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21
Q

Use only [?] that have been designed for sampling the nasopharyngeal mucosa

A

synthetic fiber swabs with thin plastic or wire shafts

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22
Q

Do not use [?], as they may contain substances that inactivate some viruses and may inhibit molecular tests

A

calcium alginate swabs or swabs with wooden shafts

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23
Q

If both NP and OP specimens are collected, combine them in a single tube to maximize [?] and limit use of [?]

A

test sensitivity

testing resources.

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24
Q

Should be [?]

A

drayon, rayon, or polyester fiber swabs

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25
Q

Select swabs with [?]

A

serrated shafts

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26
Q

Do not use [?] nor ones with [?]; they may inhibit PCR

A

calcium alginate or cotton swabs; wooden sticks

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27
Q
  1. Tilt patient’s head back [?]
  2. Insert [?] through nares parallel to palate (not upwards) until:
    a. Resistance is met, OR - Completely insert until there is resistance (about [?])
    b. Distance is equivalent to half the distance from the [?].
  3. Gently [?] the swab.
  4. Leave swab in place for [?] to absorb secretions.
  5. Slowly remove the swab while rotating it and immediately place in sterile tube containing [?].
A

70°

flexible shaft mini-tip swab

2 inches

patient’s ear to their nostril

rub and roll

several seconds

transport medium

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28
Q

Insert swab into the

A

posterior pharynx and tonsillar areas

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29
Q

Rub swab over both tonsillar pillars and posterior oropharynx and avoid touching the

A

tongue, teeth, and gums

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30
Q

Place swab, tip first, into the [?] provided.

A

transport tube

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31
Q

Store respiratory specimens at [?] after collection

A

2-8°C for up to 72 hours

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32
Q

If a delay in testing or shipping is expected, store specimens at

A

70°C or below

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33
Q

Store extracted nucleic acid samples at

A

-70°C or lower

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34
Q

allows the safe transfer of

A

viruses, chlamydia and mycoplasma, conventional cell culture methods, diagnostic tests, and molecular biology techniques

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35
Q

One of the standardized components of VTM by CDC and WHO
to prevent microbial growth:

A

o Calcium
o Magnesium
o heat-inactivated Fetal Bovine Serum (FBS)
o Antibiotics (Gentamycin and Amphotericin B)

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36
Q

and contains heat-inactivated Fetal Bovine Serum (FBS)

A

Calcium and Magnesium

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37
Q

: growth supplement for in vitro cell culture

A

Fetal Bovine Serum (FBS)

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38
Q

UNACCEPTABLE SPECIMENS
[?] specimens (specimen container info and form do not match)
No identification on [?]
Specimens with [?] for testing
Improper specimen type sent
Spillage or possibility of [?]
Specimens shipped at [?]
Excessive specimen [?]

A

Improperly identified

specimen container or form

insufficient Quantity

cross contamination

improper temperature

transport time

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39
Q

One of the most critical part in molecular techniques because downstream procedures and analysis relies on the quality of the DNA used for the assay

A

DNA ISOLATION

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40
Q

Refers to the process of separating DNA from other cellular materials such as proteins and membranes.

A

DNA ISOLATION

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41
Q

Removes potential inhibitors to the polymerase chain reaction PCR) amplification and produces a stable solution of highquality DNA that can be stored for prolonged durations without degrading

A

DNA ISOLATION

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42
Q

SOURCES OF DNA

A

Plasmid DNA
Viral Nucleic Acids
Genomic DNA from Blood and Biological Fluids
Genomic DNA from Tissue and Cells
Genomic DNA from Forensic Samples
Genomic DNA from Plant and Fungi
Genomic DNA from Food and Feed
Ancient DNA (archeologic)

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43
Q

3 BASIC STEPS

A

01 LYSIS
02 PRECIPITATION
03 PURIFICATION

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44
Q

The cell and the nucleus are broken open to release the DNA inside.

A

01 LYSIS

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45
Q

separates DNA from the cellular debris.

A

02 PRECIPITATION

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46
Q

Extracted DNA can be rinsed with alcohol to remove any remaining unwanted material and cellular debris.

A

03 PURIFICATION

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47
Q

At this point the purified DNA is usually redissolved in water for easy handling and storage.

A

03 PURIFICATION

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48
Q

LYSIS METHODS

A
  1. Mechanical disruption
  2. Using the following detergents and enzymes:
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49
Q
  • can be a manual method using mortar and pestle, homogenizer
A
  1. Mechanical disruption
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50
Q
  1. Using the following detergents and enzymes:
A

Organic: NaOH, Sodium Dodecyl Sulfate

Inorganic: Tris-EDTA (TE), EDTA, SDS

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51
Q

PRECIPITATION METHODS

A
  1. Na+ ions
  2. Ethanol/ Isopropanol
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52
Q

is added and causes DNA to precipitate out of the aqueous solution because it is not soluble in alcohol

A
  1. Ethanol/ Isopropanol
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53
Q

neutralize the negative charges on the DNA molecules which makes them more stable and less water soluble.

A

Na+ ions

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54
Q
  • normally, DNA has a
A

(-) charge

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55
Q

: to precipitate and prevent dissolving

A
  • less water soluble
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56
Q
  • dehydrants
A

Ethanol/ Isopropanol

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57
Q
  • also causes DNA to precipitate out of the aqueous solution
A

Ethanol/ Isopropanol

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58
Q
  • to separate DNA and precipitate
A

Ethanol/ Isopropanol

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59
Q
  • to purify or clean further with an alcohol or buffer
A

PURIFICATION

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60
Q
  • redissolve and resuspend in water
A

PURIFICATION

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61
Q

REAGENTS USED FOR IDENTIFICATION

A
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62
Q

designed to lyse outer cell membrane, but will not break down nuclear membrane

A

CELL LYSIS BUFFER

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63
Q

Nucleus is intact

A

CELL LYSIS BUFFER

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64
Q

remove contaminating protein

A

PHENOL

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65
Q

allows protein to settle at the bottom

A

PHENOL

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66
Q

prevents cutting of DNA during isolation.

A

CHLOROFORM

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67
Q

It solubilizes lipids and a lot of protein to remove them from the DNA

A

CHLOROFORM

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68
Q

Comes with phenol

A

CHLOROFORM

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69
Q

Contaminating proteins and lipids also settles at the bottom

A

CHLOROFORM

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70
Q

DNA floats at the top of the solution

A

CHLOROFORM

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71
Q

remove most of the protein by digesting with proteolytic enzymes

A

PROTEINASE K

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72
Q

Active against a broad spectrum of native proteins and denatured proteins

A

PROTEINASE K

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73
Q

Alternative for phenol and chloroform

A

PROTEINASE K

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74
Q

An enzyme method

A

PROTEINASE K

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75
Q

chelating agent of d Mg2+.

A

EDTA

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76
Q

Mg2+is a cofactor for DNase if the Mg2+is bound up by

A

EDTA

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77
Q

DNAses are inactivated.

A

EDTA

78
Q

Protects the DNA from damage

A

EDTA

79
Q

Inactivates DNAses that destroys the DNA

A

EDTA

80
Q

: important cofactor of DNAse; once chelated, DNAse will not work anymore

A

Magnesium

81
Q

is an anionic detergent which helps cell membrane and nuclear envelope to break open

A

SODIUM DODECYL SULFATE (SDS)

82
Q

Inhibit RNAse and DNAse.

A

SODIUM DODECYL SULFATE (SDS)

83
Q

A lysis regant

A

SODIUM DODECYL SULFATE (SDS)

84
Q

Inactivates nucleases

A

SODIUM DODECYL SULFATE (SDS)

85
Q

Prevent DNA and RNA degradation

A

TRIS-EDTA (TE) BUFFER

86
Q

Used for final resuspension and storage because it is a buffer that preserves any changes in pH

A

TRIS-EDTA (TE) BUFFER

87
Q

Preserves DNA and RNA

A

TRIS-EDTA (TE) BUFFER

88
Q

Precipitation of DNA

A

95% ETHANOL OR ISOPROPANOL

89
Q

: organic procedure

A

Ethanol

90
Q

: inorganic procedure

A

Isopropanol and phenol

91
Q

DIFFERENT TYPES OF DNA EXTRACTION METHODS

A

1.Chemical based DNA extraction method

2.Solid phase DNA extraction method

92
Q

A.Organic solvent-based DNA extraction

A
  1. Phenol-chloroform Method
93
Q

Commonly used in the laboratory

A

Phenol-chloroform Method

94
Q

Used in the RNA isolation of covid 19 testing

A

Phenol-chloroform Method

95
Q

Advantage: Rapid denaturation of nucleases (destroys RNA and DNA) and stabilization of RNA (phenol and chloroform can be used)

A

Phenol-chloroform Method

96
Q

Drawbacks: Laborious and manually intensive processing

A

Phenol-chloroform Method

Silica Column-based DNA Extraction Method

97
Q

Phenol-chloroform Method Steps:

A
98
Q

(open it up to free the nucleus; addition of buffer/detergent/enzyme)

A

Lyse/Homogenize cells

99
Q

: addition of phenol and chloroform then centrifuge

A

Extraction

100
Q

: organic phase separated

A

Precipitation

101
Q

: aqueous soln with precipitated DNA

A

Top

102
Q

: Protein contaminants and cellular debris

A

Bottom

103
Q

: Proteins (separate by placing to another container → add water → clean it further by washing with buffer or alcohol → resuspend with water)

A

Middle

104
Q

Precipitated DNA will be purified or rehydrated or resuspended with water or buffer

A

Phenol-chloroform Method

105
Q

Phenol + chloroform

A

Phenol-chloroform Method

106
Q

is good for stabilization

A

Chloroform

107
Q

Lipids and cell membrane lipids will dissolve in [?] since it is an organic compound

A

phenol

108
Q

After release of DNA from the cell, further purification requires removal of contaminating proteins, lipids, carbohydrates, and cell debris through

A

high salt, low pH and phenol & chloroform

109
Q

: extracting reagents

A

phenol & chloroform

110
Q

: increases the yield and purity of DNA

A

high salt, low pH

111
Q

: DNA will be pipeted for precipitation with ethanol

A

Supernatant

112
Q

: Phenol and Chloroform will extract the proteins

A

Bottom

113
Q

: added to prevent RNA contamination

A

RNAse

114
Q

B. Inorganic solvent-based DNA extraction

A
  1. Salting out Method
  2. Proteinase K Method/ Enzymatic Method
115
Q

Salting out Method Steps:

A

Precipitation

116
Q

Salting out Method

Precipitation: [?] helps in the DNA extraction

A

Isopropanol and salts ( sodium acetate, sodium chloride, potassium acetate and ammonium acetate)

117
Q

facilitates high DNA yield but it is time-consuming.

A

Proteinase K Method/ Enzymatic Method

118
Q

Good in research, experimentation, forensic

A

Proteinase K Method/ Enzymatic Method

119
Q

Extract as much DNA

A

Proteinase K Method/ Enzymatic Method

120
Q

Also if not maintained well in a cold chain, the (?) cannot be utilized for a longer period of time

A

Proteinase K Method/ Enzymatic Method

121
Q

The lower stability of the enzyme is another major issue in this method.

A

Proteinase K Method/ Enzymatic Method

122
Q

Disadvantage: Labile

A

Proteinase K Method/ Enzymatic Method

123
Q

Solid phase DNA extraction method

A

A. Silica column based DNA extraction method

124
Q

: (+) charged; has affinity for DNA to isolate it

A

Silica column/Silica beads

125
Q

: Rapid denaturation of nucleases and stabilization of RNA

A

Silica column based DNA extraction method

126
Q

: Laborious and manually intensive processing

A

Silica column based DNA extraction method

127
Q

Difficult method

A

Silica column based DNA extraction method

128
Q

Principle: works on the unique chemistry interaction between silica and DNA. A positively charged particles (column/bids) bind/ adsorbed with the negatively charged DNA during centrifugation

A

Silica column based DNA extraction method

129
Q

Silica column based DNA extraction method

Blue:
Black:
Droplet:

A

Blue: silica
Black: DNA
Droplet: elite DNA

130
Q

Silica column based DNA extraction method
1. Lyse cells and [?] to remove large cell debris
2. Apply [?] to silica column (spin column)/ beads
3. Addition of [?] (extraction rgt) and acidification to remove contaminating
4. [?]/ Nucleic acid extractor machine
5. Adsorbed DNA will be washed using [?]
6. Final product [?] (purified DNA; extraction of adsorbed DNA)

A

spin

lysate reagent

alcohol

Microcentrifugation

buffers

Elute DNA

131
Q

Widely accepted because of its good quality DNA yield and
minimal simple operating system.

A

Silica column based DNA extraction method

132
Q

The method is most rapid, reliable, accurate and consumes less time.

A

Silica column based DNA extraction method

133
Q

This extraction lasts up to 30 mins

A

Silica column based DNA extraction method

134
Q

RNA Extractor Machine

A

Silica column based DNA extraction method

135
Q

is a cation-chelating resin (positively-charged) binds to the negatively charged phosphate of DNA and helps in the extraction of DNA

A

Chelex

136
Q

rapid and used in forensic

A

DNA extraction using the anionic resins

137
Q

Positively charged magnetic beads attract the negatively charged DNA.

A

DND extraction by magnetic beads

138
Q

The DNA is separated under the magnetic field.

A

DND extraction by magnetic beads

139
Q

is used to fractionate DNA based on density

A

CsCI Density gradient method

140
Q

A procedure for separating particles (such as viruses or ribosomes or molecules such as DNA ) in which the sample is placed on a preformed gradient such as sucrose or caesium chloride.

A

Density gradient Centrifugation

141
Q

Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are ‘banded” in the gradient and can be collected as a pure fraction.

A

Density gradient Centrifugation

142
Q

Blood sample + sucrose or caesium chloride (ficoll: rgt)

A

Ficoll Density Gradient Centrifugation

143
Q

Source of DNA: WBC

A

Ficoll Density Gradient Centrifugation

144
Q

Nuclear DNA and Mitochondrial DNA stays

A

Ficoll Density Gradient Centrifugation

145
Q

DNA can be stored for [?] in a refrigerator or stored frozen for [?]

A

months

years

146
Q

Extracted DNA is typically stored at [?] for long-term storage, to prevent nuclease activity - To prevent DNAse from degrading the DNA

A

-20°C and up to -80°C

147
Q

are protein enzymes found in cells that degrade DNA to allow the cells to recycle nucleotide components

A

Nucleases

148
Q

Nucleases need [?] to work properly so one of the measures to prevent them from digesting DNA in blood is the use of EDTA

A

magnesium

149
Q

The [?] chelates, or binds up, most of the free magnesium and thus helps prevent the nucleases from destroying the DNA in the collected blood sample

A

EDTA

150
Q

: good for inactivating nucleases

A

EDTA

151
Q

is usually more stable over time.

A

Highly concentrated DNA

152
Q

DNA can gradually hydrolyze in

A

acidic conditions

153
Q

As such, storage buffers are slightly basic such as

A

Tris buffer

154
Q

of total RNA is ribosomal RNA

A

80-90%

155
Q

is messenger RNA

A

2.5-5%

156
Q

SARs-COV 2: [?] is extracted

A

RNA

157
Q

Difficult and very crucial since [?] are major contaminant

A

RNAse

158
Q

One of the most critical part in molecular techniques because downstream procedures and analysis relies on the quality of the DNA used for the assay

A

RNA ISOLATION

159
Q

Refers to the process of separating DNA from other cellular materials such as proteins and membranes.

A

RNA ISOLATION

160
Q

Removes potential inhibitors to the polymerase chain reaction PCR) amplification and produces a stable solution of highquality DNA that can be stored for prolonged durations without degrading

A

RNA ISOLATION

161
Q

Requires STRICT precautions to avoid sample degradation.

A

RNA ISOLATION

162
Q

RNA especially

A

labile

163
Q

Goal: Prevent RNA degradation by

A

RNAses

164
Q

is a common contaminant

A

RNAse

165
Q
  • naturally occurring
A

RNAse

166
Q
  • bacterial and human source
A

RNAse

167
Q
  • could be difficult to inactivate because it can reactivate even after autoclaving
A

RNAse

168
Q

Must be eliminated or inactivated before isolation of RNA

A

RNAse

169
Q

There must be a

A

RNAse-free

170
Q

Protecting Specimen Against RNAse:
• Wear [?] at all times
• Use [?]
• Use [?], chemicals
• Pre-treat materials with extended heat (180 C for several hours), wash with [?]
• Supplement reactions with [?]
• Reusable [?] is seldom used

A

gloves

RNase-free tubes and pipet tips

dedicated, RNase-free

DEPC-treated water, NaOH orH202

RNase inhibitors

glassware

171
Q

Common: Phenol-Chloroform Extract

A
172
Q
  1. Cell lysis step for RNA isolation is performed in detergent or phenol in the presence of [?].
  2. RNA is precipitated by addition of two volumes of ethanol or one volume of isopropanol
    a. Top:
    b. Bottom:
  3. RNA precipitate is then washed in [?] and resuspended in RNF buffer or water.
A

high salt or RNase inhibitors

aqueous solution with total RNA ; Protein contaminants solvents

70% ethanol

173
Q

is added at the lysis step or purification step to eliminate contamination of DNA

A

DNase

174
Q

is a strong denaturant of RNases and can be used instead of high-salt buffers

A

Guanidine isothiocyanate (GIC)

175
Q

A positively charged silica particles bind with the negatively charged RNA during centrifugation

A

Silica column based RNA extraction method

176
Q

mRNA: only [?] of the total RNA

A

2.5% to 5%

177
Q

Principle: All mRNAs have a [?] (many adenine)

A

poly (A) tail

178
Q

could be used to specifically capture mRNA with magnetic beads/ columns with poly T chain

A

Poly(A tail)

179
Q

[?] columns or beads bind the polyA tail of mRNA

A

Oligo polythymine (or polyuracil)

180
Q

The bead/column has a

A

3’ T 5’

181
Q

mRNA has

A

5’ A 3’

182
Q

mRN(A) is attracted to

A

T

183
Q

3 Adenine will form hydrogen bonds with

A

3 Thymine

184
Q

After washing away residual RNA, [?] is eluted by washing the column with warmed, low-salt buffer containing detergent to break the hydrogen bonds between the mRNA and the column.

A

polyA RNA

185
Q

WHY DO WE WANT TO EXTRACT ONLY THE MRNA?

Because mRNA represents the genes that are expressed and will be translated into

A

proteins

186
Q

mRNA with instructions for making the [?] is developed in a lab

A

spike protein (s-gene)

187
Q

(: lacks s-gene)

A

omicron

188
Q

mRNA enters the cell (extracted using ?)

A

poly-T bead

189
Q

COVID-19 virus [?] created

A

spike protein

190
Q

are recognized by the immune system, which produces specific antibodies against the COVID-19 virus

A

Spike proteins

191
Q

If you’re infected with the COVID-19 virus, [?] bind to virus & stop it from replacing

A

antibodies

192
Q

Proteinase K Method/ Enzymatic Method

[?] of blood + [?] TE buffer
Mix well and centrifuge at [?]
To pellet add [?] TE buffer
Mix well and centrifuge at [?]
To pellet add [?] Proteinase K
+ (?) of DNA extraction buffer
Mix well and incubate it At [?]
Add [?] + Salt: NaCI / sodium acetate
Mix well and centrifuge at [?]
Wash with [?]
Mix well and centrifuge at [?]]
Dissolving DNA in [?]

A

2ml; 20uL

2500pm, 20min

20uL

2500pm, 15min

20uL

2 ml

56°C to 60°C for 2hrs

Isoamyl alcohol

9000 rpm, 2 min

ethanol

9000pm, 2 min

TE Buffer

Molecular Biology Lec (13 decks)

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