5/16/22, 11:38 AM Molecular Diagnostics Chapter 3 Flashcards | Quizlet

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Molecular Diagnostics Chapter 3

Terms in this set (43)

Which sample types must be buccal cells, saliva, bone, teeth, hair, dried blood,
amplified prior to analysis? fixed tissue

What are two ways to break 1. homogenization through physical grinding

down cell walls for DNA 2. treatment of detergent (SDS) with strong base
extraction? (NaOH) in ethylene-diaminetetraacetic acid (EDTA)

Highly branched sugar Solution w/ specific density

that allows only RBCs and neutrophils to pass
Ficoll through
Mononuclear cells (monocytes and lymphocytes)
form a band on top

1. density-gradient centrifugation of whole blood or

bone marrow with ficoll
What are two methods for
2. incubation of whole blood or bone marrow in
separating nucleated cells?
hypotonic water which will lyse RBC's prior to
WBC's, and then centrifuged to pellet the WBC's

Which fixative is the worst for Bouin's and B-5

DNA recover?

Which fixative is the best for buffered formalin

DNA recovery?

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Molecular Diagnostics Chapter 3 in suspension using alkaline (NaOH)Study

1. lyse cells and
a detergent (SDS)
2. treat lysed debris with acetic acid and salt,
centrifuge to pellet
3. add phenol and chloroform to separate solution
Steps of Organic DNA into two layers an upper aqueous phase which
Extraction contains the DNA and a lower organic phase which
contains the cell debris ie proteins
4. precipitate DNA with ethanol, centrifuge, remove
ethanol and reprecipitate with water/buffer, can
add RNAse

What can be used to help carrier molecules such as yeast, RNA, glycogen
precipitate low amounts of
DNA?

In inorganic DNA isolation, or proteins

"salting out" procedures, in the
presence of low-pH and high-
salt concentrations, which
intracellular component
precipitates out of solution?

1. lyse suspended cells using detergent and tris

buffer
2. add high salt and low pH, sodium acetate to
precipitate protein from solution and leave DNA in
inorganic DNA isolation
solution, centrifuge
3. remove aqueous layer and precipitate DNA with
isopropanol, centrifuge, remove ethanol and add
buffer or water

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Molecular Diagnostics Chapter

How can DNA concentration
3
can be adjusted by adding or removing Study

water/buffer volume post extraction, if a low quant

be adjusted post extraction?
water is better than TE buffer

EDTA is part of TE buffer and is a chelating agent

Why his EDTA used for
that makes DNA more stable and prevents DNase
longterm DNA storage?
activity

1. cell lysis
2. acidification separating cell debris on the bottom
by centrifugation and leaving DNA in aqueous
solution
Solid Phase DNA Isolation 3. take aqueous solution of DNA and run through
column with low pH buffer to adhere to silica
column and remove solution
4. wash DNA with buffer
5. elute DNA with a low salt buffer from the column

Modern Systems of solid columns or beads

phase isolation utilize which
two matrices?

Silica and ____________ are used to high salt concentrations

bind DNA onto solid matrices

Is alkaline lysis used to isolate plasmid DNA because chromosomal DNA will be
plasmid DNA or chromosomal denatured by alkaline lysis and cannot be
DNA and why? recovered

What removes proteins during proteinase K

the extraction process?

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Molecular Diagnostics Chapter 3 centrifugation to pellet intact cells and

uses slow Study

then fast centrifugation will pellet mitochondria

from supernatant and then proceed per typical
Mitochondrial DNA isolation extraction use proteinase to remove proteins and
precipitate with cold ethanol

PCR makes it possible to replicate minimal amounts

DNA isolation techniques of DNA that can be used for downstream assays.
changed with advent of PCR. Now small amounts DNA can be extracted through
How? What are the rapid methods such as chelex beads mixed with the
advantages? sample--> lyse by boiling--> extract with
chloroform. Helpful for forensic testing

Can RNAses be eliminated by No, they can renature and regain activity
autoclaving?

Reusable glassware for RNA baked at 400 C for 4-6 hours to eliminate RNAses
isolation use must be treated
how?

What is the most abundant rRNA, 80-90%

type of RNA?

DEPC-dethyle pyrocrabonate
How to eliminate RNAse? vanadylribonucleoside complexes, macaloid clays
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What buffer cannot be used Tris, due to amine conversion mechanism of DEPC
with DEPC?

Why must RNA isolation use to inactivate intracellular RNAses

liquid nitrogen?

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Molecular Diagnostics
What type ofRNA is typically Chapter 3
mRNA, 2.5-5% Study

required for analysis? How

abundant is it?

by utilizing the polyA tail nature of mRNA. Columns

How can specifically mRNA be with polyU oligos or polyT oligos attached will grad
isolated? onto the poly A tail of mRNA. This helps to increase
yield.

Fixed tissue and solid tissue are bad, good

_________ for RNA recovery. Bone
marrow and cultured cells are
__________ for RNA recovery.

1. secondary structure of mRNA reduces the

efficiency of polyA tail and oligo binding
2. mRNA with short polyA tails may not bind to the
column
What limits good mRNA 3. A-T rich DNA fragments may bind to the polyT
recovery using columns with and poly U oligos and outcompete/contaminate the
polyT and polyA oligos? mRNA
4. cannot be treated with DNase since it will
interfere with column oligos. (except for RF DNase)
5. rRNA may also be isolated and contaminate the
mRNA elution, not pure

Which dyes bind to DNA? ethidium bromide, SyberGreen I

Which dyes bind to RNA? ethidium bromide, Sybergreen II

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Molecular
After running gelDiagnostics Chapter 3
single, double, smearing Study

electrophoresis DNA appears

as a ______________ band and RNA
appears as a __________________
band (single or double) and
without _____________ to ensure
quality.

How is quantity of DNA or RNA by using a control with a known quantity and
measured from gel comparing fluorescence intensity or densitometry
electrophoresis?

A=Ebc
A, absorbance
E, molar absorptivity (L/molcm)
b, path length (cm)
Beer-Lambert Law equation c, concentration mg/L
Conversion factor 50 ug/ml DNA per absorbance
unit, 40ug/mL per absorbance unit RNA
1 absorbance unit @ 260 nm =50 ug of DNA/mL and
40 ug of RNA/mL

Nucleic acids absorb light at 260

_________nm through adenine
residues

to determine concentration of solutions typically

What is Beer's Law used for?
DNA, RNA, protein sing absorptivity constants

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Molecular Diagnostics
A DNA preparation diluted Chapter 3
0.200 absorbance units50ug/mL per absorbance
Study

1/100 yields and absorbance unit100= 1000 ug/mL

reading of 0.200 at 260 nm.
How would you obtain the
concentration in ug/mL?

An RNA preparation diluted A. 0.500 absorbance unit40 ug/mL absorbance

1/10 velds and absorbance unit10= 200 ug/mL.
reading of 0.500 at 260 nm.
Part A What is the B. 200 ug/mL *0.2 mL= 40 ug
concentration? Part B, If the
volume of the samples was 0.2
mL what is the mass inn ug of
RNA yield?

Protein absorbs light at _____nm 280nm

at tyrosine and tryptophan
residues.

1.6 to 2.0 times more, if greater than 2.0 could have

RNA contamination, if less protein contamination.
May need to refuter through column or repeat
260/280 ratio for pure DNA
protein digestion, does not differentiate between
heavily fragmented or degraded DNA will still get a
reading

260/280 ratio for pure RNA 2.0-2.3 times more

DNA: DABA, Hoechst 33258-combines with AT bp

so specific for intact double-stranded DNA,
Which dyes are used for DNA
Picogreen-most sensitive ds DNA detection,
in fluorometry? Which are used
oligogreen- ssDNA detection
for RNA?
RNA: sybergreen II (will also bind with DNA need to
use DNase)

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Molecular Diagnostics Chapter 3

Spectrophotometry: detects through absorption,
Study

does not require calibration with known sample,

does not have time limit on read time, detects any
nucleic acid even if degraded or nicked because
will be absorbed by uv light, used for an array of
procedures, RNA may be read through absorption
Compare and Contrast
and affect results
spectrophotometry and
fluorometry
fluorometry: detects through fluorescence of dyes,
requires calibration with known sample, must be
read within two hours, detects ds DNA that is intact,
used for high sensitivity procedures like NGS, RNA
will not be detected

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