ANALYSIS OF DNA

Macaraig, Jeph Roxy M.

MBB 206 T1-L
 ANALYSIS OF DNA
WHY DO WE ANALYZE DNA?

 To check for integrity, purity, size, and concentration

 To identify genes in the genome

 To understand evolution, function, and interaction of genes

 Forensic, diagnostic, and phylogenetic applications

 Development of products with commercial applications
 ANALYSIS OF DNA
DNA ANALYSIS METHODS

I. Gel electrophoresis

II. Southern blotting

III. Sequencing
IV. Next Generation
Sequencing Technologies
GEL ELECTROPHORESIS
 GEL ELECTROPHORESIS
 Charged molecules are separated in
response to an electric field

 Uses a slab of gel that acts as a sieve

 To determine DNA integrity, size (in no. of

bp or in MW), concentration

 Restriction mapping, molecular genetic

diagnosis/ fingerprinting, fragment
separation
 GEL ELECTROPHORESIS
DNA samples placed in
wells cut in gel
Gel slab
Voltage supply

Negative electrode

Positive DNA fragments

electrode + move from negative
to positive electrode
 GEL ELECTROPHORESIS
Gel matrix is a porous material
acting as sieve.
Agarose : for wide range of DNA sizes
Polyacrylamide : for proteins and
narrow range of DNA sizes
Starch : protein electrophoresis

Buffer provides constant, uniform

electric charge.

Voltage supply attracts the

negatively charged DNA and RNA
 GEL ELECTROPHORESIS
Loading dye weighs down the sample.
10x BlueJuice™ Gel Loading Buffer (Invitrogen)
 65% (w/v) Sucrose
 10 mM Tris-HCl (pH 7.5)
 10 mM EDTA
 0.3% (w/v) Bromophenol Blue

Gel stain helps visualize the DNA in the gel

 Ethidium bromide – toxic and mutagenic
 SYBR™ Safe DNA Gel Stain (Invitrogen)
 SYBR™ Green I (Invitrogen)
 SYBR™ Gold (Invitrogen)
 GelRed (Biotium)
 GEL ELECTROPHORESIS
VISUALIZATION
Gel stains intercalates with DNA which
fluoresce under UV light

DNA ladders are used for size estimation.

 GEL ELECTROPHORESIS
+

Shorter fragments
travel faster through the
gel.

Longer fragments are

retarded in the gel.

–
 GEL ELECTROPHORESIS
FACTORS AFFECTING MIGRATION

 Size of the molecule

 Electrical charge

 Viscosity of the gel

 Voltage applied

 Amount of time voltage is applied

 GEL ELECTROPHORESIS
Different gel concentrations resolve varying
DNA fragment sizes.

Table 1. Agarose gel concentration for resolving linear DNA molecules

Adapted from Magdeldin (2012)

SOUTHERN BLOTTING
 SOUTHERN BLOTTING
 Established by Edward Mellor Southern
in 1975

 Reveals information about DNA identity

(fingerprinting), size, and abundance

 Uses separation technique based on gel

electrophoresis
SOUTHERN BLOTTING
 SOUTHERN BLOTTING
I. DNA extraction and restriction
digestion

II. Gel Electrophoresis

III. Denaturation and depurination

IV. Blotting

V. Probe labeling

VI. Hybridization and washing

VII. Detection
 SOUTHERN BLOTTING
I. DNA extraction and restriction
digestion
to isolate the gene of interest

II. Gel electrophoresis

Fragments are separated by size
 SOUTHERN BLOTTING
III. Denaturation and depurination

 DEPURINATION (0.25 N HCl)

 Creates apurinic sites that reduce
binding
 Predisposes PO4 backbone to
cleavage

 DENATURATION (NaOH)
 dsDNA ssDNA
 Sugar-phosphate backbone is
cleaved
 SOUTHERN BLOTTING
IV. Blotting

 Bands are transferred on a membrane by

capillary action
 Nitrocellulose
 Nylon

 Usually done overnight

 Transfer methods:
 Capillary
 Electroblotting
 Vacuum or positive pressure blotting

 Immobilization step for attachment

 UV cross-linking
 Baking at 120° C for 30 min.
 Baking at 80° C for 2 hrs
 SOUTHERN BLOTTING
V. Probe labeling

 ssDNA probe with sequence

homologous to target DNA

 Probe labels:
Radioactive isotopes
Non-radioactive labels
 SOUTHERN BLOTTING
V. Probe labeling

 Radioactive isotopes:

 32P, 35S, 125I, 3H

 High sensitivity (32P detects single

copy of gene in 0.5 ug of DNA)

 DISADVANTAGES:
Short half life
Radioactive waste
Access to dark room
 SOUTHERN BLOTTING
V. Probe labeling

Non-radioactive labels:

Safer to use but not as

sensitive

Biotin, digoxygenin,
enzymes, antibodies
 SOUTHERN BLOTTING
VI. Hybridization and washing

Incubation of labeled probes with

DNA fragments on membrane

Unhybridized probes are washed by

several changes of buffer
 SOUTHERN BLOTTING
VII.Detection

Depends on probe used

Radioactive labels: Autoradiography

Non-radioactive labels:
Colorimetric
Fluorescent
Chemiluminescent
SEQUENCING
 SEQUENCING
Why do we sequence DNA/ genome?
 To identify genes in the genome
 To understand evolution of genes and
genomes
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ccgtatccactccttgccctcctgataa
gattatctttgatgcagagaggggggag
tacatttgctctgaaactggagaagttt
tagaagataaaattatagatcaagggcc
agagtggagggccttcacgccagaggag
aaagaaaagagaagcagagctaggctct

 To study functions and interactions of genes

 To develop products with commercial

applications
 SEQUENCING
Sequencing technologies in 1900s:

1. Maxam-Gilbert chemical cleavage

method

Developed by Alan Maxam and

Walter Gilbert

Radioactive labeling at 5’ end of the

DNA fragment

Chemical cleaving in sequence-

dependent manner
 SEQUENCING
Sequencing technologies in 1900s:

1. Maxam-Gilbert chemical cleavage

method
 Dimethyl sulfate: Purines
 Hydrazine : Pyrimidines
 SEQUENCING
Sequencing technologies in 1900s:

2. Sanger chain termination method

 Developed by Frederick Sanger

 Enzymatic termination of DNA synthesis at
random sites using dideoxynucleotides

 For routine sequencing applications

 SEQUENCING
 Sequencing technologies in 1900s:

2. Sanger chain termination method

 Reagents used:

 ssDNA as template

 Oligonucleotide primers

 DNA polymerase

 Dideoxynucleotide triphosphates
(ddNTPs)

 Fragments are separated in 4 lanes

 Visualized by UV light
 SEQUENCING
2. Sanger chain termination method

 Development of automated
sequencer
 Uses fluorescent tags, one
for each base
 Adenine
 Thymine
 Guanine
 Cytosine

 Sequence is recorded as
chromatogram
SEQUENCING
NEXT GENERATION SEQUENCING (NGS)
TECHNOLOGIES
 NGS TECHNOLOGIES

Enabled sequencing of millions of DNA molecules simultaneously

Can be used in entire genome sequencing

High-throughput and reduced cost

 NGS TECHNOLOGIES
1. Pyrosequencing – Roche 454

2. Sequencing by synthesis – Illumina (Solexa)

3. Sequencing by ligation – SOLiD (Applied Biosystems)

4. IonTorrent™ Semiconductor sequencing (Life Technologies)

5. Real-time long-read sequencing (Oxford Nanopore Technologies®)

 NGS TECHNOLOGIES
1. PYROSEQUENCING (Roche 454)
 relies on the detection of pyrophosphate

 dsDNA are denatured into ssDNA and are captured by

beads

 Amplification by Emulsion PCR

 NGS TECHNOLOGIES
1. PYROSEQUENCING (Roche 454)

 dNTPs complement the template

bases

 PPi is released as bases are added

 PPi transformed to ATP

 Measured by chemiluminescence
NGS TECHNOLOGIES
 NGS TECHNOLOGIES
2. SEQUENCING BY SYNTHESIS (ILLUMINA-SOLEXA)

 DNA shearing and adapter ligation

 Graft into flow cell or single molecular array

 Bridge amplification to form clonal DNA fragments

 Sequencing using ddNTPs with cleavable fluorescent

dyes
NGS TECHNOLOGIES
 NGS TECHNOLOGIES
3. SEQUENCING BY LIGATION (SOLID- APPLIED BIOSYSTEMS)
 Sequencing by Oligo Ligation Detection

 DNA is sheared and adapters are ligated

 Hybridization to beads

 Amplification by emPCR

 Beads attached to glass slides

 Fluorescent dye-labeled probes for
sequencing
 NGS TECHNOLOGIES
3. SEQUENCING BY LIGATION (SOLID- APPLIED BIOSYSTEMS)
NGS TECHNOLOGIES
 NGS TECHNOLOGIES
4. IONTORRENT™ SEMICONDUCTOR SEQUENCING (LIFE TECHNOLOGIES)

 Hydrogen ions are released as

nucleotides are added.

 pH change detected by ion sensor

NGS TECHNOLOGIES
 NGS TECHNOLOGIES
5. REAL-TIME LONG-READ SEQUENCING (OXFORD NANOPORE TECHNOLOGIES®)

 DNA is initially fragmented to 8–10 kb.

 A leader and a hairpin adapters are ligated to either

end of the fragmented dsDNA.

 Adapters direct dsDNA through the pore

 Voltage shifts as DNA passes through the pore

NGS TECHNOLOGIES
 NGS TECHNOLOGIES
Table 2. Advantages and mechanisms of sequencers

Adapted from Liu, et. al. (2012)

 NGS TECHNOLOGIES
Table 3. Applications of sequencers

Adapted from Liu, et. al. (2012)

 NGS TECHNOLOGIES
Table 4. Comparison of Ion Torrent and Oxford Nanopore sequencing technologies

Sequencing Cost per Gb

Read length Throughput Runtime Error profile
platform (US $)

1 %,
Ion Torrent 200-400 bp 50 Mb – 15 Gb 2 – 7 hrs 25-3,500
insertion/deletion

~12 % insertion/
Oxford Nanopore ~200 kb 1.5 Gb – 4 Tb Up to 48 hrs 750
deletion

Adapted from Goodwin, et. al. (2016)

REFERENCES
 REFERENCES
Behjati, S & PS Tarpey. (2013). What is next generation sequencing?. Arch Dis Child Educ Pract Ed. 2013;98:236–238. doi:10.1136/archdischild-
2013-304340

Brown, TA. (2001). Southern Blotting and Related DNA Detection Techniques. eLS. DOI: 10.1038/npg.els.0000996

Dingman, CW, MP Fisher, and T Kakefuda. (1972). Role of molecular conformation in determining the electrophoretic properties of
polynucleotides in agarose-acrylamide gels. II. Biochem. 11, 1242 – 1250.

Goodwin, S, JD McPherson, WR McCombie. (2016). Coming of age: ten years of next-generation sequencing technologies. Nature Reviews:
Genetics 2016; 17:333-351

Liu, L, Y Li, S Li, N Hu, Y He, R Pong, D Lin, L Lu, M Law. (2012). Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and
Biotechnology Volume 2012, Article ID 251364

Magdeldin, S. (2012). Gel Electrophoresis – Principles and Basics. InTech, Rijeka, Croatia. ISBN 978-953-51-0458-2

Ravi, I, M Baunthiyal, J Saxena (eds). Advances in Biotechnology. Springer India 2014. ISBN 978-81-322-1554-7 (eBook) DOI 10.1007/978-81-
322-1554-7

Wall, WJ. (2002). Techniques for DNA Analysis. Ullmann's Encyclopedia of Industrial Chemistry. DOI: 10.1002/14356007.e26_e01

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THANK YOU! 
ANALYSIS OF DNA
Macaraig, Jeph Roxy M.
MBB 206