High Quality Genomic DNA:

Choosing the best extraction method

(Parte 2)

Dra. Pamela Leal

Pamela.leal@ufrontera.cl

12/09/2024 APA478 Clase 4

Journal of Biorepository Science for Applied Medicine 2014:2
1) LYSIS BUFFER:
• Sodium dodecyl sulfate (SDS) and TritonTM X-100 are used to
solubilize cell membranes.

• Proteinase K is used to cleave glycoproteins and inactivate RNases

and DNases.

• Urea, guanidinium salts, and chemical chaotropes have been used

to disrupt cells and inactivate cellular enzymes.

2) DNA PRECIPITATION:
• by adding high concentrations of salt, such as ammonium acetate
to counteract repulsion caused by the negative charge of the
phosphate backbone.

• Ethanol (final concentrations of 70%–80%)

• Isopropanol (final concentrations of 40%–50%)

3) DNA ELUTION:
• TE buffer is commonly used for long-term DNA storage because it
prevents it from being damaged by nucleases, inadequate pH,
heavy metals, and oxidation by free radicals. Tris provides a safe pH
of 7–8.

• EDTA chelates divalent ions used in nuclease activity and

counteracts oxidative damage from heavy metals.
 Genomic DNA Extraction by Sample Type
Virus

Tissue

Cells

Blood

Plant

Body fluids

Bacterial

Forensics
 Genomic DNA Extraction by DNA Type

Cell-Free DNA

FFPE Tissue

Low-Input Samples

Not FFPE-Compatible

Single Cells

Plasmids

PCR products
Genomic DNA Extraction by application and technologies

Next-generation
✓ 16s rRNA Sequencing
Sequencing
✓ Amplicon Sequencing
✓ ChIP-Seq
✓ Custom Sequencing
PCR ✓ De Novo Sequencing
✓ Exome Sequencing
✓ Genotyping by Sequencing
✓ Long-Read Sequencing
✓ Methylation Sequencing
DNA cloning ✓ miRNA and Small RNA Sequencing
✓ mRNA Sequencing
✓ Shotgun Sequencing
✓ Target Enrichment
DNA sequencing ✓ Targeted DNA Sequencing
✓ Targeted RNA Sequencing
✓ Whole-Genome Sequencing
✓ Whole-Transcriptome Sequencing
DNA
electrophoresis
 Input DNA - how much is needed by application?

Whole- genome microarray analysis continues to require an input DNA mass that is at
least 100 times larger than that required for simple PCR testing and requires very pure
DNA that is double stranded with a length span at least 5 times longer than required for
most PCR reactions

However, when other panels For whole genome de novo

A DNA quantity of 2.5 to 3.0
and techniques are used for sequencing, a very high DNA
mg is necessary depending on
whole genome genotyping, a quantity is required, usually
the array size and platform
higher quantity of DNA, up to from 30 to 60 mg depending
used
6.0 mg is required on the platform.

For whole genome sequencing, For targeted resequencing of A minimum concentration of

usually a quantity above 10 custom regions of interest a 50 ng/ml is also necessary in
mg, ideally 20 mg DNA is lower DNA quantity of about 3 both microarray and NGS
desirable to 6 mg is used analysis.
 DNA/RNA isolation considerations for genomic

Others are introduced

through the sample
Humic/fulvic acid in plant treatment or extraction
samples method: EDTA, heparin,
or phenol:chloroform.
Hemoglobin in blood

EDTA

A wide variety of DNA and RNA sample types and extraction methods can introduce inhibitors
to enzymatic reactions.
What inhibitors are
found in different
sample types?
What inhibitors are found in different sample
treatments/extraction methods?

https://support.illumina.com/bulletins/2016/05/dnarna-isolation-considerations-when-using-truseq-library-prep-kits.html
Purification and isolation technologies

https://assets.thermofisher.com/TFS-Assets/BID/brochures/dna-isolation-purification-brochure.pdf
Organic extraction
DNAzolTM Reagent / DNAzol BD Reagent / Plant DNAzol Reagent

Uses a guanidine detergent lysing solution that hydrolyzes RNA and

allows the selective precipitation of DNA from the lysate.
 It is the method of
choice when large
This procedure is
amounts of mammalian Approximately 200 µg of
derived from a method
DNA are required mammalian DNA, 100- The usual yield of DNA
originally described by
(Southern blotting or for 150 kb in length, is from 20 ml of normal
Daryl Stafford and
construction of genomic obtained from 5 x 10⁷ blood is ~ 250 µg.
colleagues (Blin and
libraries in culture aneuploid cells.
Stafford 1976).
bacteriophage λ
vectors).
Column purification
InvitrogenTM PureLinkTM Genomic DNA Mini Kit

Utilizes solid phase extraction techniques to bind and isolate DNA

within columns containing silica or glass fiber filter membranes.

✓ Samples are digested using an optimized buffer that aids in

protein denaturation and enhances Proteinase K activity.
✓ RNase A is added to remove any residual RNA, and the sample
is lysed using a InvitrogenTM PureLinkTM lysis and binding
buffer.
✓ The lysate is then mixed with ethanol to precipitate the gDNA.

Downstream apllications

• Cloning
• PCR,
• NGS
• Genotyping
• Southern blotting
DNA column purification kit selection guide
Magnetic beads
https://www.youtube.com/watch?v=hrKM7d6_8hQ

https://www.youtube.com/watch?v=ukZp6Adu534

https://www.youtube.com/watch?v=v5KFe43b2Xk
 High-throughput 96 Well Purification Kits
PRODUCT
PRODUCT PRODUCT NO. YIELD SPEED STARTING MATERIAL TECHNOLOGY APPLICATIONS
DESCRIPTION
200 µL fresh blood,
GenElute 96 Well frozen blood or Rapid isolation of Silica membrane PCR, restriction
BLD9601
Blood Genomic DNA 4 to 6 µg per well 70 min/plate blood treated with genomic DNA from with vacuum or spin digestion, cloning
BLD9604
Purification Kit EDTA, citrate, or whole blood format and southern blots
heparin
Rapid isolation of
Less than 20 mg
GenElute 96 Well DNA from human Silica membrane PCR, restriction
G1N9602 tissue or less than
Tissue Genomic DNA 12 to 15 µg per well 70 min/plate and animal tissue, with vacuum or spin digestion, cloning
G1N9604 10µL cultured cells
Purification Kit cultured cells and format and southern blots
or bacteris
bacteria
Capillary
Complete removal Silica membrane sequencing,
GenElute 96 Well PCR9601 Less than 100 µL
75-95% recovery 45 min/plate of primers and with vacuum or spin Microarray analysis,
PCR Clean-up Kit PCR9604 PCR reaction
primer-dimers format ligation and
restriction digestion
Transfections
(robust cells,
GenElute HP 96- Rapid purification of Silica membrane
1.3 mL bacterial sequencing,
Well Plasmid NA9604 Up to 10 µg 45 min/plate transfection grade with vacuum or spin
culture per well restriction
Miniprep Kit plasmid format
digestion, cloning
and PCR

Less than Purification of total

GenElute 96 Well Silica membrane RT-PCR, northern
RTN9602 107 cultured cells, RNA from animal or
Total RNA 50 to 200 ng/µL 70 min/plate with vacuum or spin blots and
RTN9604 less than 30 mg himan cell cultures
Purification Kit format microarray analysis
tissue or saliva and tissue

https://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/ht-96-well-purification-
Specialty kits for DNA purification
https://www.thermofisher.com/cl/es/home/life-science/dna-rna-purification-
analysis/protocol-videos.html
 High Quality Genomic DNA:
Choosing the best extraction method
(Parte 2)

Dra. Pamela Leal

Pamela.leal@ufrontera.cl

27/09/2021 APA478 Clase 4

Measure the concentration of the DNA/RNA
 How is the optical signal generated?

Photometry Fluorescence
The photometric measurement of The fluorometric measurement
nucleic acids is based on the intrinsic of nucleic acids is based upon
absorptivity properties of nucleic acids the use of fluorogenic dyes that
at 260 nm. bind selectively to DNA or RNA.
 How is the optical signal measured?

Photometry Fluorescence

The signal is measured by The signal is measured by

spectrophotometers. The attenuation fluorometers. Sample is excited with
in the light that reaches the detector filtered light (at the excitation
after passing through the sample is wavelength, and the emitted light (at
measured in relation to the incident the emission wavelength) is recorded
light and expressed as absorbance by a detector.
values of the sample in the solution.

Wavelength separation can take place Wavelength separation can take place
before or after the light has passed the in various ways (for example with
sample, and the optical light path can filters or with monochromators)
be horizontal or vertical.
 How is the concentration calculated?

Photometry Fluorescence

Concentrations of nucleic acids can Concentrations are measured using

be directly calculated from their the fluorescence signal of the
measured absorbance values at 260 sample and a calibration curve is
nm, using the Beer-Lambert's generated from standard samples
equation: of known concentration and fit to
appropriate regression models.
C= A
εL

C= nucleic acid concentration in molar (M)

A=UV absorbance in absorbance units (AU)
ε=wavelength-dependent molar absorptivity
coefficient in M-1cm-1
L= light path in cm

This concentration calculation is The limit of detection and linear

automated in many instruments. response of the measurements
are specific to each assay.
What are the advantages and disadvantages?
Choice instruments for photometric UV-Vis RNA/DNA
quantification

Drop (1 μL) Drop (1 μL) 96-384 well plates 6-384 well plates
cuvettes µDrop Plate (2-10 μL) µDrop Plate (2-10 µL)
Choice instruments for fluorescence RNA/DNA quantification

1-20 μL 1-20 μL
What is the best way to determine the quality/purity
of my DNA?

• The ratio A260/A280 is typically used to measure purity of the sample.

• For DNA, the ideal ratio is 1.8, but could be in the range 1.7–1.9.
• The A260/A230 ratio is also used to determine if contamination is present. The ideal
ratio for DNA is 1.8–2.0.
• Purity of DNA can also be examined by gel analysis. For genomic DNA, look for high
average fragment size.
Valores indicativos de pureza en muestras de ADN:

https://www.bancoadn.org/docs/programa-control-calidad-muestras.pdf
https://www.bancoadn.org/docs/programa-control-calidad-muestras.pdf
https://international.neb.com
 High Quality Genomic DNA:
Choosing the best extraction method
(Parte 2)

Dra. Pamela Leal

Pamela.leal@ufrontera.cl

09/2024 APA478 Clase 4