Summary Advanced Molecular Biology
Molecular techniques
Primary vs immortalized vs induced pluripotent stem cells
Absolute vs relative measurement
- Absolute → how many molecules are present
per cell/sample
- Relative → how much more/less molecules are
present in different cells/samples
- Most molecular techniques that analyze specific
gene/mRNA/proteins measure relative
abundance (e.g. treated vs untreated, wt vs
mutant)
- Absolute measurement requires standards with known quantities/concentration (e.g. cDNA
for qRT-PCR)
Accuracy vs precision
- Accuracy → how close the
measurement is to the true or
acceptable value
- Precision → how close
measurements of the same item are to each other
- Absolute measurement should be both accurate and precise
- Relative measurements do not necessarily need to be accurate as far as the control is
suffering from the same bias/inaccuracy
Measurement of one specific DNA/RNA
- Quantitative PCR (qPCR)
o RFU (relative fluorescent unit) indicates the relative amount of double stranded DNA
in the reaction via incorporation of dsDNA-specific fluorescent dye (e.g. SYBR green)
1
, o The number of cycles needed to reach the log phase of
amplification (threshold cycle number) is proportional
to the number of starting molecules present in the
sample
o Melting curve of the amplified DNA enables testing if
the desired product is amplified (i.e. specificity of the
PCR)
o mRNAs need to be converted to cDNA in a reverse
transcription first (RT-qPCR)
- Southern and Northern blots
o Southern blot detects specific DNA
sequences in a (genomic) DNA sample
digested with restriction enzymes and
separated on agarose gel
o Northern blot detects specific messenger or
micro RNA in total RNA samples separated
on a denaturing agarose gel
o Specific DNA/RNA pieces are detected by
radioactively labeled DNA probes
complementary to the sequences
o Specificity of the detection is defined by
stringency of hybridization and washing
steps
Measurement of one specific protein
- Western blot
o Western blot detects specific proteins in a
sample separated based on their relative mass
(~size) on polyacrylamide gel under denaturing
condition (SDS-PAGE)
o After separation, proteins are transferred to
and immobilized on a membrane
o The membrane is blocked by protein
containing solution (‘milk’ or BSA) to prevent
nonspecific binding
o Specific proteins or posttranslational
modifications (e.g. phosphorylation) are
detected by primary antibody, which in turn is detected by
secondary antibody conjugated to enzymes (e.g. HRP) to
enable visualization
o Specificity of the detection is defined by specificity of the
antibody and the stringency of washing steps
Measurement of ‘all’ mRNA/(c)DNA in a sample
- Microarray
o Microarray can detect relative abundance of cDNA multiple
proteins in parallel
2
, o For microarray analysis mRNA or cDNA from different samples needs to be labelled
with different fluorescent dye (typically red and green)
o Microarray is limited to the analysis of genes/mRNAs for which complementary DNA
probes is present on the array
o The specificity of the analysis is defined by the uniqueness of the probe sequence
and the stringency of the hybridization and washing steps
- NGS
o NGS enables parallel sequencing of
DNA or cDNA fragments from millions
of fragments and hence quantification
of all DNA/mRNA present in a sample
o The most commonly used Illumina
technology required incorporation of
specific adaptor sequences to each
fragment, which are then used for
solid phase amplification of millions of
separate clusters of DNA fragments on
a glass surface covered by adapter
specific primers (the so-called ‘flow
cell’)
o These individual clusters (composed of
identical DNA sequences) are then
subjected to sequencing using
fluorescently labelled nucleotides and
imaging of the clusters after
incorporation of each nucleotide
o NGS enables highly specific and
quantitative detection and hence
is in many ways superior to
microarray analysis
Measurement of ‘all’ proteins in a sample
- Mass-spectrometry
o MS can detect (peptides from)
multiple proteins in parallel
o For proteome analysis, proteins
need to be digested to
‘measurable-sized’ peptides
(typically by trypsin), which can
be fractionated to less complex
mixture via liquid
chromatography (LC)
o Electrospray is most commonly
used to detect ratio of the mass
(m) of charged molecule (a
molecular ion) to its charge (z), or
m/z (MS spectra). This in turn can
3
, be used to infer the absolute mass and most likely amino acid composition of a
peptide
o In an MS/MS instrument such as the ion trap, a specific peptide ion can be selected
for fragmentation into smaller ions that are then analyzed (MS/MS spectra) to infer
the order of amino acids within the peptide
o MS can detect proteins with high specificity, but measuring relative or absolute
quantities of proteins require extra tricks or specialized instruments
Sorting cells based on total DNA/RNA content or presence of specific proteins
- Fluorescence activated cell sorting
o FACS enables analysis and/or sorting of
cells based on light scattering and/or
fluorescent signal
o Enables detection and sorting of specific
cell types (e.g. T cells from blood) within a
population using fluorescently-labelled
antibody-based detection of cell-type-
specific cell surface antigens
o Enables detection of different DNA
content in cells using fluorescent DNA dyes
(e.g. DAPI, Hoechst, 33342) and hence
separation of cells based on for example
ploidy level or cell cycle stage
o Enables sorting of cells that express (a
combination of) different fluorescent
proteins
Cellular sub-fractionation
- Differential or density gradient centrifugation
o Different cellular components and
organelles have different sedimentation
constants and density and hence can be
separated by centrifugation
o The purity of different fractions can be
assessed by antibodies against
compartment specific proteins (e.g.
histones – chromatin/nuclei or cell
surface receptors – membrane, etc)
o The obtained cellular fraction can then
be used to assess localization of a
specific protein (Western blot) or define
the proteasome of a
compartment/organelle (MS)
4
Molecular techniques
Primary vs immortalized vs induced pluripotent stem cells
Absolute vs relative measurement
- Absolute → how many molecules are present
per cell/sample
- Relative → how much more/less molecules are
present in different cells/samples
- Most molecular techniques that analyze specific
gene/mRNA/proteins measure relative
abundance (e.g. treated vs untreated, wt vs
mutant)
- Absolute measurement requires standards with known quantities/concentration (e.g. cDNA
for qRT-PCR)
Accuracy vs precision
- Accuracy → how close the
measurement is to the true or
acceptable value
- Precision → how close
measurements of the same item are to each other
- Absolute measurement should be both accurate and precise
- Relative measurements do not necessarily need to be accurate as far as the control is
suffering from the same bias/inaccuracy
Measurement of one specific DNA/RNA
- Quantitative PCR (qPCR)
o RFU (relative fluorescent unit) indicates the relative amount of double stranded DNA
in the reaction via incorporation of dsDNA-specific fluorescent dye (e.g. SYBR green)
1
, o The number of cycles needed to reach the log phase of
amplification (threshold cycle number) is proportional
to the number of starting molecules present in the
sample
o Melting curve of the amplified DNA enables testing if
the desired product is amplified (i.e. specificity of the
PCR)
o mRNAs need to be converted to cDNA in a reverse
transcription first (RT-qPCR)
- Southern and Northern blots
o Southern blot detects specific DNA
sequences in a (genomic) DNA sample
digested with restriction enzymes and
separated on agarose gel
o Northern blot detects specific messenger or
micro RNA in total RNA samples separated
on a denaturing agarose gel
o Specific DNA/RNA pieces are detected by
radioactively labeled DNA probes
complementary to the sequences
o Specificity of the detection is defined by
stringency of hybridization and washing
steps
Measurement of one specific protein
- Western blot
o Western blot detects specific proteins in a
sample separated based on their relative mass
(~size) on polyacrylamide gel under denaturing
condition (SDS-PAGE)
o After separation, proteins are transferred to
and immobilized on a membrane
o The membrane is blocked by protein
containing solution (‘milk’ or BSA) to prevent
nonspecific binding
o Specific proteins or posttranslational
modifications (e.g. phosphorylation) are
detected by primary antibody, which in turn is detected by
secondary antibody conjugated to enzymes (e.g. HRP) to
enable visualization
o Specificity of the detection is defined by specificity of the
antibody and the stringency of washing steps
Measurement of ‘all’ mRNA/(c)DNA in a sample
- Microarray
o Microarray can detect relative abundance of cDNA multiple
proteins in parallel
2
, o For microarray analysis mRNA or cDNA from different samples needs to be labelled
with different fluorescent dye (typically red and green)
o Microarray is limited to the analysis of genes/mRNAs for which complementary DNA
probes is present on the array
o The specificity of the analysis is defined by the uniqueness of the probe sequence
and the stringency of the hybridization and washing steps
- NGS
o NGS enables parallel sequencing of
DNA or cDNA fragments from millions
of fragments and hence quantification
of all DNA/mRNA present in a sample
o The most commonly used Illumina
technology required incorporation of
specific adaptor sequences to each
fragment, which are then used for
solid phase amplification of millions of
separate clusters of DNA fragments on
a glass surface covered by adapter
specific primers (the so-called ‘flow
cell’)
o These individual clusters (composed of
identical DNA sequences) are then
subjected to sequencing using
fluorescently labelled nucleotides and
imaging of the clusters after
incorporation of each nucleotide
o NGS enables highly specific and
quantitative detection and hence
is in many ways superior to
microarray analysis
Measurement of ‘all’ proteins in a sample
- Mass-spectrometry
o MS can detect (peptides from)
multiple proteins in parallel
o For proteome analysis, proteins
need to be digested to
‘measurable-sized’ peptides
(typically by trypsin), which can
be fractionated to less complex
mixture via liquid
chromatography (LC)
o Electrospray is most commonly
used to detect ratio of the mass
(m) of charged molecule (a
molecular ion) to its charge (z), or
m/z (MS spectra). This in turn can
3
, be used to infer the absolute mass and most likely amino acid composition of a
peptide
o In an MS/MS instrument such as the ion trap, a specific peptide ion can be selected
for fragmentation into smaller ions that are then analyzed (MS/MS spectra) to infer
the order of amino acids within the peptide
o MS can detect proteins with high specificity, but measuring relative or absolute
quantities of proteins require extra tricks or specialized instruments
Sorting cells based on total DNA/RNA content or presence of specific proteins
- Fluorescence activated cell sorting
o FACS enables analysis and/or sorting of
cells based on light scattering and/or
fluorescent signal
o Enables detection and sorting of specific
cell types (e.g. T cells from blood) within a
population using fluorescently-labelled
antibody-based detection of cell-type-
specific cell surface antigens
o Enables detection of different DNA
content in cells using fluorescent DNA dyes
(e.g. DAPI, Hoechst, 33342) and hence
separation of cells based on for example
ploidy level or cell cycle stage
o Enables sorting of cells that express (a
combination of) different fluorescent
proteins
Cellular sub-fractionation
- Differential or density gradient centrifugation
o Different cellular components and
organelles have different sedimentation
constants and density and hence can be
separated by centrifugation
o The purity of different fractions can be
assessed by antibodies against
compartment specific proteins (e.g.
histones – chromatin/nuclei or cell
surface receptors – membrane, etc)
o The obtained cellular fraction can then
be used to assess localization of a
specific protein (Western blot) or define
the proteasome of a
compartment/organelle (MS)
4
