MSC IV SEMESTER_DNA Profiling - DNA quantification.pdf

DNA Profiling - DNA quantification

1. DNA Profiling

2. DNA QUANTIFICATION
Historical and commonly
used quantitation
methods include the
following:
•Yield gels
•Spectrophotometry
•Fluorometry
•Slot blot hybridization
•AluQuant®
•Quantitative PCR (qPCR)

3. Yield Gels
✓semi-quantitative/qualitative assay
✓The method consists of theelectrophoresisof
DNA in an agarose gel matrixincorporating a
fluorescentintercalating dyesuch as ethidium
bromide (EtBr).
✓The concentration of a sample can be
determined by comparing the intensity of the
fluorescence of the sample to that of the
calibration standards.
✓Larger (undegraded) DNA fragments migrate
at a slower rate than those of lower molecular
weight.
✓Degraded DNA will consist of lower molecular
weight fragments.
https://nij.ojp.gov/nij-hosted-online-training-courses/dna-extraction-and-
quantitation-forensic-analysts/quantitation/yield-gels

5. Absorption Spectrophotometry
•Theoptical density (O.D.)of a solution
containing 50 µg/mL of double-stranded
DNA or 40 µg/µl of single-stranded
DNA is 1.00 at a wavelength of 260nm.
•The quality or purity of the sample can
be determined by comparing the
measurements at 260 and at 280 nm
(the wavelengths for which DNA and
protein absorb)

8. Fluorometry
•the molecules lose some of the absorbed
energyby irradiating light of a longer
wavelength. This property is known as
fluorescence.
•There are several dyes : fluorescence
enhancement when bound to double-
stranded DNA.
•PicoGreen
®
is one of the more common
fluorescent dyes that can be used in this
way.
•The fluorescence is easily measured using
a fluorometer.

9. Fluorometry

11. Slot Blot Hybridization
•The majority of laboratories used the
commercially availableQuantiBlot
®
kit,
which employs the following procedures:
Extracted DNA is
denatured to single-
stranded DNA is bound to
a positively charged nylon
membrane
a probe complementary to
the D17Z1 locus (present
in high quantities in higher
primates) is applied and
allowed to hybridize to
the DNA.
hybridized complex is
detection i.e
colorimetry/Chemiluminesc
ence,

12. Detection methods include:
•Colorimetry: tetramethylbenzidine
(TMB), which yields a blue color when
oxidized by hydrogen peroxide.
•Chemiluminescence: ACES(Gibco BRL)
and(Amersham Biosciences). The
chemiluminescent reactions cause the
release of photons that are captured on
film or a digital imaging device.
•Chemiluminescence is more sensitive
than colorimetry and can detect down to
10 to 20 pgof DNA

14. AluQuant
•Alu sequences are abundant in the human genome numbering approximately
•Alu repeats (500,000 to 1,000,000 copies per genome. )were probed in the AluQuant
Human DNA Quantitation System.
denaturing the sample
and incubating it with the
AluQuantEnzyme and
AluQProbe solutions
production of adenosine
triphosphate (ATP)
(correlated with the
amount of DNA present)
ATP was determined by
using a luminometer

16. Reference
https://nij.ojp.gov/nij-hosted-online-training-courses

17. This Photo by Unknown Author is licensed under CC BY-SA