WEEK 8 – MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY Flashcards
o Chemical added in the solution to remove RNAses
o Inhibits RNAses
o Degrades nucleic acid
DIETHYL PYROCARBONATE (DEPC)
o Precipitating agents
iSOPROPANOL and ETHANOL
o Lysing reagents
TRITON X-100, CTAB, SDS
- agents used
most frequently for visualization of bands after electrophoresis are __ and_.
o Both of these types of dyes specifically associate with
nucleic acid.
DETECTION SYSTEMS
- FLUORESCENT DYES and SILVER STAIN
HOW TO ENSURE THAT THE EXTRACTED SAMPLE IS
OF GOOD QUALITY?
o fluorescent dye and silver stain ___
o Fluorescent dye ,,,
-> Used for DNA and RNA
o__ -> used for proteins
- add detection systems
- Ethidium bromide, gel red, sybr green, sybr gold
- Silver stain
Analyze DNA and RNA for quality
Fluorescent dyes are used in this procedure
1
2
Electrophoresis
o Ethidium bromide
o SybrGreen
DNA bands migrate towards _ DNA is _ charged
anodic area
negatively
negative electrode
Cathodic Area
positive electrode
Anodic Area
Basis of separation the more DNA is larger, the more
they will be found on the _ area. The more the DNA
is smaller, the more they will be found on the _ area
cathodic
anodic
powerful toll to visualize bands
UV Transilluminator
* Appearance depends on type of DNA isolated
o __ should collect as a
bright band with low mobility
o A high-quality preparation of RNA will yield two distinct band
of __
o The integrity of these bands is an indication of the integrity of
the other RNA species present in the same sample
Electrophoresis
High molecular-weight genomic DNA
rRNA
Alternatively, enclosed gel systems contain __ inside a plastic-enclosed gel cassette, limiting exposure and limiting waste.
- carcinogenic
Therefore, DNA separated in _ or _ and exposed to it will emit _ when illuminated at 300 nm.
EtBr
- agarose or acrylamide
- orange light
After soaking or running in dye, the DNA illuminated with UV light will appear as _ in the gel.
The image is captured with appropriate filters by digital transfer to analytical
software.
orange bands
Minor Groove–Binding Dyes
is one of a set of
stains introduced in 1995 as another type of
nucleic acid–specific dye system.
SYBR green ( N ′ , N ′ -dimethyl-N -[4-[(E)-(3methyl-1,3-benzothiazol-2-ylidene)methyl]
1-phenylquinolin-1-ium-2-yl]-N
propylpropane-1,3-diamine)
used for double-stranded DNA
staining;
differs from EtBr in that it
does not intercalate between bases; it sits in the minor groove of the double helix.
- in association with DNA or
RNA also emits light in the orange range at _
SYBR green I
- (522 nm)
for single-stranded DNA or RNA
staining;
SYBR green II,
one of these agents and was the most widely used dye in
early DNA and RNA analyses.
ETHIDIUM BROMIDE
(3,8-diamino-5-ethyl-6-
phenylphenanthridinium bromide – EtBr)
- carcinogenic, precautions are required to limit exposure.
Under excitation with UV light at 300 nm, EtBr in DNA
emits visible light at _
▪ UV excitation nanometer – <350
▪ Visible nanometer – _
▪ Infrared –_
▪ DNA is found at_
590 nm
350-700
>700
UV spectrum
DNA separated in agarose or acrylamide and exposed to EtBr will emit _ when illuminated at _
ORANGE light
300 nm
for both DNA and RNA staining.
SYBR gold,
MINOR GROOVE-BINDING DYES is Not commonly used because it requires optical filters
EtBr – _
SYBR green – _
SYBR gold – _
590nm
522nm
537nm
i n agarose gel electrophoresis, __is 25-100 times more sensitive than EtBr (detection level: _ pg of double stranded DNA versus_ for EtBr)
SYBR staining
60pg
5 ng
cyanine dye of proprietary structure.
Its fluorescence increases more
than 1,000-fold upon binding to
double-or single-stranded DNA
or to RNA.
SYBR gold stain
like SYBR green, _ is excited by UV light (300-nm
wavelength) and emits light at _.
SYBR gold
537 nm
is the preferred dye for real-time PCR methods.
SYBR green
Another sensitive staining system originally developed for protein visualization.
After electrophoresis, the sample is fixed with __ and __
The gel is then impregnated with__ and __ in a weakly acid solution.
It is especially useful for protein analysis and for detection of limiting amounts of product
SILVER STAIN
- methanol and
acetic acid
- ammoniacal silver (silver
diamine) solutions or silver nitrate
a SILVER STAIN particular/specific for dying adenine and thymine bonds
HOECHST DYE
Sample absorbance are determined on the spectrophotometer
at _ and _
o Nucleic acid (DNA, RNA, Nucleotides) absorb light at
_
o Protein _ ;_
o_ measure of DNA purity
SPECTROPHOTOMETRY
- 260nm and 280nm
- 260nm
- 280nm; 230nm
- A260/A280 ratio
SPECTROPHOTOMETRY absorbance wavelength is _ to the concentration of nucleic acid
o used to measure spectral purity of DNA and RNA
Using _, concentration can be determined from the absorptivity constants
Constant OD unit
o _ DNA
o _ RNA
DIRECTLY PROPORTIONAL
BEER-LAMBERT LAW
50
40
LAMBERT’S LAW
concentration of a substance is
directly proportional to the absorbed light, and inversely
proportional to the amount of transmitted light.
A= abc
o a = molar absorptivity (constant: 6.23)
o b = light path
o c = concentration of substance
DETERMINING CONCENTRATION
Using the absorptivity as a conversion factor from Optical
Density to Concentration
o _ – nucleic acids
o _ – proteins
260
280
DETERMINING CONCENTRATION
Formula
Concentration = 0D260nm x OD unit x Dilution Factor
Whereas:
o OD260nm – Spectrophotometer reading
o OD unit – Absorbance Unit (50 – DNA, 40 – RNA)
o Dilution Factor – 1:10, 1:100.
One Optical Density (OD) unit or absorbance unit at 260 nm is
equal to
o 50 μg/mL for DNA
o 40 μg/mL for RNA
DETERMINING PURITY
OD260nm should be _ more than OD280nm
1.6-2.00 times
- 𝑶𝑫𝟐𝟔𝟎𝒏𝒎 ÷ 𝑶𝑫𝟐𝟖𝟎𝒏𝒎 = OD Ratio
MEASUREMENT OF OPTICAL PURITY
o DNA _
o RNA _
1.8 (low limit 1.6) (range: 1.6 to 2.00)
2.0
If the OD ratio is less than_ for DNA, _ for RNA, this
indicates contamination
o Usually with __
1.6
2.0-2.3
protein
DNA – If the OD ratio is higher than 2.0, it may be
contaminated with _
Ratio of the reading:
o _ measure of purity
RNA
𝑶𝑫𝟐𝟔𝟎𝒏𝒎 ÷ 𝑶𝑫𝟐𝟖𝟎𝒏𝒎
Pure preparation of DNA and RNA have OD Ratio of
o DNA – 1.8
o RNA – 2.0
utilizes fluorescent dyes which specifically bind DNA or RNA
- Measures fluorescence related to DNA concentration in
association with DNA-specific fluorescent dyes
Fluorometry (or Fluorescence Spectroscopy)
are relatively specific to nucleic acids as opposed to protein and other cellular components
it increases when they bind
nucleic acids
fluorescent dyes
It requires a negative control (to set the zero point on the
fluorometer) and a standard of known concentration
Fluorometry (or Fluorescence Spectroscopy)
FLUOROMETRY uses probe or dye __
-> If DNA/RNA is absent – it will not bind
FLUOROPHORE
– sensitive analytical method that determines
presence or absence of DNA
o Consist of_ and _
Fluorometry
primary and secondary monochromators
About 1000 times more sensitive than spectrophotometric
absorbance
* Less susceptible to protein and RNA contamination
FLUOROMETRY
Does not give a crude measurement of purity
* Does not assure that the DNA or RNA is not degraded
* Frosted part of the glass interferes with the detection of
fluorescent light when using glass cuvettes in the fluorometer
FLUOROMETRY
many laboratories continue to use the EtBr due to the requirement for __for detection of sybr green emission at __
wavelengths and SYBR gold at_
New instrumentation with more flexible detection systems allows
utilization of the SYBR and other fluorescent stains.
special optical filters
520-to 550-nm
537 nm
