In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA. You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.

1. QUANTITATION
OF
DEOXYRIBONUCLEIC
ACID
(DNA)

2. TABLE OF CONTENTS
 Absorption Spectrophotometry
 Yield Gel Method
 Slot Blot Technique
References
DNA Quantitation Meaning
Purpose of DNA Quantitation
Methods Used for the Quantitation of DNA

3. DNA QUANTITATION MEANING
 In simple terms quantitation means to determine and measure the quantity of
anything measurable.
 DNA quantitation means quantifying or measuring the useful and required
concentration of DNA present in the Sample.

4. PURPOSE OF DNA QUANTITATION
 The primary purpose of DNA quantitation in forensic casework is to determine
the appropriate amount of DNA template to include in PCR amplification of short
tandem repeat loci in order to avoid off-scale data and associated artefacts.
 A PCR amplification reaction may fail due to the presence of co-extracted
inhibitors, highly degraded DNA, insufficient DNA quantity, or a combination of
all of these factors.
 Therefore the reason behind performing a DNA quantification test is to
determine the amount of “amplifiable” DNA present in the sample.

5. Illustration of STR typing results at a single heterozygous locus for a
single source sample with (a) too much DNA template showing off-
scale, split peaks, (b) too little DNA template where the arrow points to
allele dropout due to stochastic effects, or (c) just the right amount so
that two allele peaks are balanced and on-scale.

6. Flow chart illustrating the role of DNA Quantitation
after the Extraction of DNA

7. METHODS USED FOR THE QUANTITATION OF
DNA
Yield Gel Method
Slot Blot Technique
Absorption Spectrophotometry

8. ABSORPTION SPECTROPHOTOMETRY
Absorption of light by DNA molecule is maximum at 260 nm. This characteristic
of DNA is used to quantitate DNA present in a solution.
 A cuvette-based spectrophotometer has a horizontal light path where the
wavelength-specific light is perpendicular to the sample. Most standard cuvettes
have fixed optical path lengths of 1 cm.
A microplate spectrophotometer, measures samples in a microplate, therefore
the light path is vertical and varies according to the volume in the microplate well.

9. Comparison of the fixed 1 cm path length of light in a cuvette based
system and the variable vertical light path of a microplate based
system

10. In Absorption Spectrophotometry the
determination of concentration and
purity of DNA in based on Beer and
Lambert’s Law.
 Purity of nucleic acid samples is
determined by finding the ratio of the
nucleic acid measurement at 260 nm and
the protein measurement at 280 nm,
where protein absorbance peaks. To
correct for background turbidity, a
measurement at about 320 nm can also
be taken, since protein and nucleic acids
do not absorb light at this wavelength.
Typical absorbance spectrum for
DNA, RNA and protein, indicating
the peak at about 260 nm for DNA
and RNA and the peak at about 280
nm for protein.

11. Approximate purity based on
A260/A280 ratio.

12. YIELD GEL METHOD
 It is basically the Agarose Gel Electrophoresis method.
The principle of quantification of DNA molecules includes
the fact that the higher the amount of DNA molecules, the more
the intensity of bands visualized under UV light. Thus, when a
standard concentration of DNA molecule is run on agarose gel
side by side with the unknown DNA fragment, by comparing
the intensity of the bands, the relative as well as absolute
quantification of the DNA molecule can be carried out.
 1% agarose gel is prepared by dissolving the agarose in
running buffer (preferably 1X Tris-acetic acid-EDTA buffer).
Intercalating dye such as ethidium bromide (EtBr) is added to
the gel.

13. Further the extracted DNA along with an indicator dye like bromophenol blue is
poured into the wells of the agarose gel, and the gel is subjected to electrophoresis
in submerged condition.
Migration pattern of charged molecules under the influence of electric field

14. The DNA molecule forms a complex with the
intercalating dye.
 The DNA-intercalating dye complex emits
fluorescence when exposed to ultraviolet light.
 The fluorescence is viewed in the shape of
fluorescence bands. The thickness of the band is co-
related with the quantity of DNA present in the
medium.
The concentration of DNA can be calculated on
comparison basis with known quantity of DNA
sample running in the same gel in another well.
Further the pattern of migration also indicates
about the quality of the DNA.

15. Different fragments of DNA in a typical DNA size marker along with
their corresponding concentration of DNA

16. SLOT BLOT TECHNIQUE
 The most commonly used method in forensic labs
during the late 1990s and beginning years of the twenty-
first century for genomic DNA quantitation was the so-
called “slot blot” procedure.
This test was specific for human and other primate DNA
due to a 40 base pair probe that bound to a region on
chromosome 17 called D17Z1.
 Slot blots involved the capture of genomic DNA on a
nylon membrane followed by addition of a human-specific
probe. Nylon membranes have a greater mechanical
strength, a higher binding capacity for nucleic acids, and a
stronger retention of bound nucleic acids.

17.  Positively charged nylon membranes provide an ionic
interaction between the negatively charged phosphate
groups of the nucleic acid and the positively charged groups
of the membrane
Chemiluminescent or colorimetric signal intensities were
then compared between a set of standards and the samples
The slot blot assay took several hours to perform and
could detect both single-stranded and double-stranded DNA
down to levels of approximately 150 pg (Walsh et al. 1992).
 Degraded DNA can be detected using this method
because the probe is a 40-mer and will therefore hybridize to
small fragments of DNA.

18. Illustration of a human DNA quantitation result with the slot
blot procedure. A serial dilution of a human DNA standard is
run on either side of the slot blot membrane for comparison
purposes. The quantity of each of the unknown samples is
estimated by visual comparison to the calibration standards.
For example, the sample indicated by the arrow is closest in
appearance to the
2.5 ng standard

20. REFERENCES
 Butler J.M. 2011. Advanced Topics in Forensic DNA Typing:
Methodology. Academic Press. P. 49-53
https://www.biotek.com/assets/tech_resources/Nucleic_Acid_Quant_
Application_Guide.pdf
 https://link.springer.com/protocol/10.1007/978-1-0716-0274-4_15