Techniques in molecular biology
To understand what is going on in a cell several techniques are required.
Select the best experimental system
o Consider which organism you experiment on (model organisms are useful to mimic
human diseases but the same findings in animals do not capture the human
situation).
o Culturing human cells (primary human cells, immortalized human cells or induced
pluripotent cells.
Primary hepatocyte human cells: limited life span and genetically difficult to
manipulate >> quickly dedifferentiate and loose cellular identity
o Immortalized human cells: immortal, can be manipulated but their cancerous cells
have little resemblance to hepatocytes.
o Induced pluripotent cells (iPS): derived from differentiated cell and can be
propagated, amendable to manipulate, derived from hepatocyte like cells that can
mimic the in vivo situation better than cell lines. But they are difficult to create.
o You could also consider 3D cell cultures over 2D cultures. Like organoids and tissue-
like structures to better resemble the human body.
Measure the amount of RNA/DNA/protein
Absolute or relative manner
- Absolute: how many molecules are present. Requires a standard with known
concentration/quantities. Like measuring cDNA for qRR-PCR
- Relative: how much more/less molecules are present in different cells/samples. Usually
molecular techniques analyse the relative abundance. (treated vs. untreated, wild type vs.
mutant.
The amount can be measured accurately or more precise.
- Accuracy: how close is a measurement to the true or accepted value.
- Precision: how close are measurements of the same item to each other.
- Absolute measurements have to be both accurate and precise.
- Relative measurements do not need to be both accurate so long as the control has the same
bias/inaccuracy.
Techniques
- Quantitative PCR (qPCR): absolute specific amounts, used to measure the amount of a
specific DNA or RNA sequence in a sample. Uses a fluorescent dye that binds to double-
stranded DNA. This allows detection of amplification in real time. The cycle number is
inversesly proportional to the initial amount of target nucleic acid.
- Northern/southern blotting: relative specific amounts, southern blot detects specific DNA
sequences, northern blot detects specific RNA sequences. Both methods involve gel
electrophoresis to separate the nucleic acids. Transfer to a membrane and hybridization with
labelled DNA probes that are complementary to the target sequence.
- Western blotting: measure specific proteins in a sample. Detects different proteins that have
been separated by size, using SDS-PAGE. Proteins are transferred to a membrane, blocked
and detected using primary antibodies.
- Microarray: used to measure all mRNA/©DNA in a sample. Measures the expression levels of
many genes at once by hybridizing labelled cDNA from different samples onto a chip
containing specific DNA probes. Relative abundance is detected by fluorescence.
- Next generation sequencing: measure all mRNA/cDNA in a sample. It allows for the
sequencing of millions of DNA or cDNA fragments in parallel.
- Mass-spectrometry: technique to measure all proteins in a sample. It can detect peptides
from multiple proteins in parallel. The proteins are cut into measurable sizes.
, - Fluorescence activated cell sorting: to sort cells based on total DNA/RNA content or
presence of specific protein. It enables the sorting based on light scattering and/or the
fluorescent signal.
Define spatial and temporal distribution
- Differential or density gradient centrifugation: to find the proteins in cellular subfractions.
Used to separate different cellular components or organelles based on their size, mass and
density.
- Fluorescent in situ hybridization: to find cellular DNA probes in a cell culture. DNA FISH uses
fluorescently labelled DNA probes to detect specific DNA sequences.
- RNA fluorescent in situ hybridization: to find cellular mRNA in a cell. RNA FISH uses
fluorescently labelled DNA probes to detect specific mRNA molecules.
- Immunofluorescence assay (IFA): to find different kinds of proteins in a tissue. Uses
antibodies. A primary antibody binds to the target protein and a fluorescently labelled
secondary antibody binds to the primary antibody.
- Live cell imaging: the solution to not have to kill the cells.
Analyses of molecular interactions between proteins or nucleic acids
- Electrophoretic mobility shift assay (EMSA): to detect the DNA-RNA interactions. Labelled
DNA or RNA fragment is incubated with a protein. If binding occurs, the resulting complex
moves more slowly through a gel than the free nucleic acid, creating a shift in position.
- Chromatin immunoprecipitation or RNA immunoprecipitation: to measure DNA-RNA
interactions. ChIP and RIP are used to study interactions between proteins and nucleic acids
in living cells. In ChIP, DNA-protein interactions are fixed with crosslinking. The DNA is
sheared into fragments, and then protein-DNA complexes are immunoprecipitated using a
specific antibody. After reversing the crosslinks, the associated DNA is analysed to identify
binding sites.
- Coimmunoprecipitation (Co-IP): to measure protein interactions. Used to identify partners of
the protein of interest or by confirming predicted interaction.
- Fluorescent resonance energy transfer (FRET): enables real life detection of proximity of two
fluorescently labelled proteins. Energy from a donor fluorophore is transferred to an acceptor
fluorophore only when they are in close proximity and proper orientation. By using excitation
wavelength of one of the fluorescent proteins and the detection emission from the other
one, one can obtain the signal from part of the cells/tissue where these two proteins tagged
by these fluorophores interact.
Investigate the consequences of functional perturbations
Mutations can cause loss of function or gain of function.
- Loss of function: causes the complete or partial loss of the gene function. Usually requires
mutation of both alleles.
- Gain of function: causes an increase in protein production of a completely new function of a
single allele is sufficient to the phenotype.
- Random mutation: mutation that occurs by chance.
- Targeted mutagenesis: modification of a specific genetic locus or gene product. Is used in
reverse genetic approach.
- Homologous recombination based gene deletion: used to delete genes. Homologous
recombination-based gene deletion.
- CRISPR-Cas9 based genome editing: used to edit genes. Is allows scientists to precisely alter
DNA within living organisms. It uses a guide RNA to target a specific DNA sequence, and the
Cas9 enzyme acts like molecular scissors to cut the DNA at that location.
To understand what is going on in a cell several techniques are required.
Select the best experimental system
o Consider which organism you experiment on (model organisms are useful to mimic
human diseases but the same findings in animals do not capture the human
situation).
o Culturing human cells (primary human cells, immortalized human cells or induced
pluripotent cells.
Primary hepatocyte human cells: limited life span and genetically difficult to
manipulate >> quickly dedifferentiate and loose cellular identity
o Immortalized human cells: immortal, can be manipulated but their cancerous cells
have little resemblance to hepatocytes.
o Induced pluripotent cells (iPS): derived from differentiated cell and can be
propagated, amendable to manipulate, derived from hepatocyte like cells that can
mimic the in vivo situation better than cell lines. But they are difficult to create.
o You could also consider 3D cell cultures over 2D cultures. Like organoids and tissue-
like structures to better resemble the human body.
Measure the amount of RNA/DNA/protein
Absolute or relative manner
- Absolute: how many molecules are present. Requires a standard with known
concentration/quantities. Like measuring cDNA for qRR-PCR
- Relative: how much more/less molecules are present in different cells/samples. Usually
molecular techniques analyse the relative abundance. (treated vs. untreated, wild type vs.
mutant.
The amount can be measured accurately or more precise.
- Accuracy: how close is a measurement to the true or accepted value.
- Precision: how close are measurements of the same item to each other.
- Absolute measurements have to be both accurate and precise.
- Relative measurements do not need to be both accurate so long as the control has the same
bias/inaccuracy.
Techniques
- Quantitative PCR (qPCR): absolute specific amounts, used to measure the amount of a
specific DNA or RNA sequence in a sample. Uses a fluorescent dye that binds to double-
stranded DNA. This allows detection of amplification in real time. The cycle number is
inversesly proportional to the initial amount of target nucleic acid.
- Northern/southern blotting: relative specific amounts, southern blot detects specific DNA
sequences, northern blot detects specific RNA sequences. Both methods involve gel
electrophoresis to separate the nucleic acids. Transfer to a membrane and hybridization with
labelled DNA probes that are complementary to the target sequence.
- Western blotting: measure specific proteins in a sample. Detects different proteins that have
been separated by size, using SDS-PAGE. Proteins are transferred to a membrane, blocked
and detected using primary antibodies.
- Microarray: used to measure all mRNA/©DNA in a sample. Measures the expression levels of
many genes at once by hybridizing labelled cDNA from different samples onto a chip
containing specific DNA probes. Relative abundance is detected by fluorescence.
- Next generation sequencing: measure all mRNA/cDNA in a sample. It allows for the
sequencing of millions of DNA or cDNA fragments in parallel.
- Mass-spectrometry: technique to measure all proteins in a sample. It can detect peptides
from multiple proteins in parallel. The proteins are cut into measurable sizes.
, - Fluorescence activated cell sorting: to sort cells based on total DNA/RNA content or
presence of specific protein. It enables the sorting based on light scattering and/or the
fluorescent signal.
Define spatial and temporal distribution
- Differential or density gradient centrifugation: to find the proteins in cellular subfractions.
Used to separate different cellular components or organelles based on their size, mass and
density.
- Fluorescent in situ hybridization: to find cellular DNA probes in a cell culture. DNA FISH uses
fluorescently labelled DNA probes to detect specific DNA sequences.
- RNA fluorescent in situ hybridization: to find cellular mRNA in a cell. RNA FISH uses
fluorescently labelled DNA probes to detect specific mRNA molecules.
- Immunofluorescence assay (IFA): to find different kinds of proteins in a tissue. Uses
antibodies. A primary antibody binds to the target protein and a fluorescently labelled
secondary antibody binds to the primary antibody.
- Live cell imaging: the solution to not have to kill the cells.
Analyses of molecular interactions between proteins or nucleic acids
- Electrophoretic mobility shift assay (EMSA): to detect the DNA-RNA interactions. Labelled
DNA or RNA fragment is incubated with a protein. If binding occurs, the resulting complex
moves more slowly through a gel than the free nucleic acid, creating a shift in position.
- Chromatin immunoprecipitation or RNA immunoprecipitation: to measure DNA-RNA
interactions. ChIP and RIP are used to study interactions between proteins and nucleic acids
in living cells. In ChIP, DNA-protein interactions are fixed with crosslinking. The DNA is
sheared into fragments, and then protein-DNA complexes are immunoprecipitated using a
specific antibody. After reversing the crosslinks, the associated DNA is analysed to identify
binding sites.
- Coimmunoprecipitation (Co-IP): to measure protein interactions. Used to identify partners of
the protein of interest or by confirming predicted interaction.
- Fluorescent resonance energy transfer (FRET): enables real life detection of proximity of two
fluorescently labelled proteins. Energy from a donor fluorophore is transferred to an acceptor
fluorophore only when they are in close proximity and proper orientation. By using excitation
wavelength of one of the fluorescent proteins and the detection emission from the other
one, one can obtain the signal from part of the cells/tissue where these two proteins tagged
by these fluorophores interact.
Investigate the consequences of functional perturbations
Mutations can cause loss of function or gain of function.
- Loss of function: causes the complete or partial loss of the gene function. Usually requires
mutation of both alleles.
- Gain of function: causes an increase in protein production of a completely new function of a
single allele is sufficient to the phenotype.
- Random mutation: mutation that occurs by chance.
- Targeted mutagenesis: modification of a specific genetic locus or gene product. Is used in
reverse genetic approach.
- Homologous recombination based gene deletion: used to delete genes. Homologous
recombination-based gene deletion.
- CRISPR-Cas9 based genome editing: used to edit genes. Is allows scientists to precisely alter
DNA within living organisms. It uses a guide RNA to target a specific DNA sequence, and the
Cas9 enzyme acts like molecular scissors to cut the DNA at that location.
