2013-05-13
1
New Developments in Capillary
Electrophoresis with focus on
Bioanalysis
Lecture 9
Christian Nilsson
Today
• CE of nucleic acids
• Electrophoretic preconcentration techniques
CE and Nucleic acids
• Separation and sequencing
– Gene analysis
• Detection of mutations
– Disease diagnostics
DNA separation
• Use of linear hydrophilic polymers for:
– Separation
– To prevent adsorption to the capillary wall
– To supress the EOF
DNA separation
• Poly(N, N-dimethylacrylamide) (PDMA)
– Used as linear polymer for DNA separation
• An alternative is to use star-shaped PDMA
polymers
– Lower viscosity compared to a linear polymer with
the same molecular weight
– Shorter separation time
– Maintained resolution
J Chromatogr A, 2011, 1218, 3037-3041
DNA separation
• Linear polymers
– Relatively high concentration necessary
• High viscosity
– High viscosity reduces separation speed and limits
capillary refreshing
– Viscosity depends on polymer size
– Controlling the polymer structure to reduce
viscosity
J Chromatogr A, 2011, 1218, 3037-3041
2013-05-13
2
Separation of DNA
• Capillary wall coating can be avoided as PDMA
adsorb to the capillary wall and reduce the
interactions between the sample and capillary
wall
CE for detection of SNP
• SNP = Single nucleotide polymorphism
• A single nucleotide is changed in the genome
• If the SNP is present in a gene for the protein,
an amino acid can change
• Examples of CE applications
CE for detection of SNP
• Different types of mutations
– Deletion: Can be separated based on length
– SNPs: More complicated as the length are the
same
CE for detection of SNP
• γ-glutamyl hydrolase gene (GGH)
– Important for methotrexate (MTX) metabolism
– MTX-(Glu)n → MTX
– SNP 452 C>T lower the activity of GGH and MTX(Glu)n
is accumulated
Electrophoresis, 2011, 32, 2021-2027
CE for detection of SNP
Electrophoresis, 2011, 32, 2021-2027
CE for detection of SNP
• Mutation in the gene for epidermal growth
factor receptor (EGFR)
• To predict the outcome of lung cancer therapy
– Therapy designed to block the EGFR by targeting
its tyrosine kinase domain
– CE can be used to detect mutations in the tyrosine
kinase domain of EGFR
• Before using an inhibitor
J Sep Sci 2010, 33, 2349-2355
2013-05-13
3
Epidermal growth factor receptor
• Start a signalling cascade when activated
• Activation of tyrosine kinase
• Important for
– DNA synthesis
– Cell division
– Involved in the immune system
CE for detection of SNP
• Most frequent mutation is a deletion
– 10-20 base pairs are deleted
• Focus on the detection of the substitution
mutation
J Sep Sci 2010, 33, 2349-2355
CE for detection of SNP
• Commercial instruments with 8, 16 or 96
capillaries were used
• The EGFR gene was amplified by PCR
J Sep Sci 2010, 33, 2349-2355
CE for detection of SNP
J Sep Sci 2010, 33, 2349-2355
Determination of methylated DNA
• Methylation in eukaryotic DNA
– Only 5-methylcytosine?
– Suggested that methyladenine is present in
eukaryotic cells but its function is unknown
Determination of methylated DNA
• DNA methyl transferase catalyse the reaction
• Cytosine methylation in eukaryotes is
associated with gene silencing
• CE to get a deeper understanding of the
biological functions of methylations
2013-05-13
4
Determination of methylated DNA
• CE-LIF has been proved to be significantly faster
and more sensitive than more traditional method
such as HPLC-UV
• The DNA is hydrolyzed prior to fluorescent
labeling
• A fluorescent dye is used for detection
– Fluorescent dye: BODIPY FL EDA
– Bind covalently to the phosphate group of the
nucleotide
– Attomol level of detection
Electrophoresis 2010, 31, 3548-3551
Determination of methylated DNA
Electrophoresis 2010, 31, 3548-3551
Analysis of methylated adenine from bacterias
Micelle to solvent stacking
• New online sample concentration technique
for charged analytes
J Chromatogr A 2010, 1217, 6290-6295
Micelle to solvent stacking
• The micelles and analytes are opposite
charged
– Cationic micelles
– Separation of anions
– Methanol as organic solvent
Micelle to solvent stacking
• In sample: Micelles + Samples
• In electrolyte: Organic solvent
• In the sample plug the analyte bind to the
micelles
• When the micelles reach the micelle to
solvent boundrary the affinity of the analytes
for the micelles will be significantly lower due
to the organic solvent
Micelle to solvent stacking
J Chromatogr A 2010, 1217, 6290-6295
10-fold
enhancement of
the sensitivity
Later more than
100-fold
enhancement of
detection has
been achived
[J Chromatogr A, 2010,
1217, 175-178]
2013-05-13
5
Sweeping combined with micelle to
solvent stacking
• For separation of organic anions
• Generally micelle to solvent stacking can be
used for either only anions or only cations
J Chromatogr 2011, 1218, 1004-1010
Sweeping combined with micelle to
solvent stacking
• Positively charged capillary coating
• Electrolyte with organic solvent
• 1. Injection of micelle solution of cationic
solvent
• 2. Injection of sample
J Chromatogr 2011, 1218, 1004-1010
Sweeping combined with micelle to
solvent stacking
J Chromatogr 2011, 1218, 1004-1010
2-3 times
improvement
compared to
micelle to
solvent
stacking
alone
Sweeping combined with micelle to
solvent stacking
J Chromatogr 2011, 1218, 1004-1010