Methods in molecular genetics 1 Flashcards
DNA/RNA extraction and purification, Quantification, separation, detection and sequencing
Summary
5 Methods and Techniques used in Molecular Genetics:
DNA/RNA extraction and purification
DNA/RNA quantification
DNA/RNA separation
DNA/RNA identification
DNA/RNA sequencing
Nucleic acids quantification
Spectrophotometry
Nucleic acids separation
Chromatography
Elecrophoresis
Nucleic acids detection and identification
Blotting
Arrays
Nucleic acids sequencing
Termination (Sanger) sequencing
Next Generation sequencing
Nucleic acid isolation and purification
Tissue disruption
Grinding tissues in a mortar
Needs to be under liquid nitrogen N2
OR
Using lysozyme to destroy bacterial cell wall
Nucleic acid isolation and purification
Tissue homogenization
Buffer systems: used to solubilise and protect
nucleic acids.
Nucleic acid isolation and purification
2 points
- Differential (phase) extraction
Uses a combination of aqueous and organic solvents to separate nucleic acids from other substances. - Ion exchange cartridges
Use charged resins that retain nucleic acids
which are eluted after all the contaminants
are washed. [DNA, RNA negatively charged. Silica positive charge = retain DNA and RNA]
-> analysis
Nucleic acid quantification
What is Spectrophotometry
4 points
- light absorbance
- Nucleic acids absorb light at 260nm
- Extinction coefficient (EC) at 260 nm: Light absorbance (optic density) in 1 ml solution in 1cm path.
e. g. Double stranded DNA: 50 (1OD: 50 ug/ul) - UV light through cuvette -> Optic Density
Nucleic acid quantification
Spectrophotometry allows
3 points
- To calculate the amount/concentration of nucleic acids (DNA, RNA)
OD x EC = x ug/ul
- To study the secondary structure of DNA. Pairing of single stranded DNA.
- To check purity of DNA and RNA.
Determined by the A260/280 ratio: OD(260nm)/OD(280nm)
OD (260): nucleic acids
OD (280): proteins
Nucleic acid separation
What is Electrophoresis
3 points
- the motion of charged particles into fluids
under the influence of an electric field. - DNA and RNA are separated based on their size
- DNA and RNA are uniformly negatively charged.
They move towards the anode (+).
Nucleic acid separation
3 types of electrophoresis
- Horizontal agarose gel-electrophoresis
Separation of DNA and RNA using Agarose gels.
- Vertical polyacrylamide gel-electrophoresis (PAGE)
Uses slide polyacrylamide gels for resolving
DNA and RNA fragments.
Higher resolution: one base pair difference
- Capillary electrophoresis
The separating gel is contained within a thin capillary.
DNA or RNA molecules are driven through
the gel under a very high voltage: 30 kV.
The capillary (gel) can be used hundreds of times.
Nucleic acid separation
Ion-exchange chromatography
4 points
- Separates molecules based on their charge.
- The charged molecules are first retained on a resin having opposite charge to them.
- All non retained molecules are washed through.
- The retained molecules are then eluted using a salt solution.
Nucleic acid separation
Size-exclusion chromatography
- Separates molecules based on their size.
- The small molecules are retained in the resin particles.
- The large molecules pass through between the resin particles.
DNA and RNA detection
4 points
- Fluorescent intercalating agents:
Ethidium bromide: orange fluorescence under UV light.
SYBRGreen: green fluorescence under UV light.
- Dyes: Hoechst, silver
- Radioactive labelling, 32P(ATP)
- Fluorescent labelling: Cy3, Cy5, 6-FAM, Rox, TAMRA, etc
DNA/RNA sequence detection and identification
What are blotting techniques
5 points
- techniques by which a specific DNA or RNA sequence is detected/identified in a complex sample.
- DNA or RNA are first separated by gel electrophoresis.
- DNA or RNA are transferred from the gel onto a synthetic membrane (nitrocellulose or nylon) using capillarity- or electro-transfer.
- Target DNA is then detected using a labelled probe which is incubated with the membrane and let to hybridize to the target sequence.
- The membrane is then washed to remove non hybridized probe then exposed to an X-ray film to detect the hybridized probe.
DNA/RNA sequence detection and identification
blotting techniques summary
4 points
- DNA separation by gel-electrophoresis
- DNA transfer onto a membrane
- The membrane is incubated with a labelled probe
- The membrane is exposed to an X-ray film
DNA/RNA sequence detection and identification
southern vs northern vs western blotting
3 points
- southern: detection of DNA sequences
probe usually DNA sequence
- Northern: detection of RNA sequences
probe usually DNA sequence
- Western: detection of proteins using antibodies
DNA/RNA sequence detection and identification
Gene chips (gene arrays)
4 points
- A collection of DNA spots arrayed on a solid matrix. Each spot contains
a probe for a gene. - The chips are hybridized with DNA or RNA samples for detection and quantification of particular sequences.
- Macro-chips:
Contain hundreds of macroscopic spots (probes) on glass or membrane supports. - Micro-chips, microarrays:
Contain thousands of microscopic spots (probes) on glass supports.
DNA sequencing
Sanger & Coulson method
3 points
- Sequencing reaction:
Single stranded DNA to be sequenced
A primer is required
dNTPs (dATP, dCTP, dGTP, dTTP) required.
Labelled ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) (big dye terminator) required.
DNA polymerase I
- Electrophoresis:
PAGE, capillary electrophoresis - Fragment detection –> sequence
High throughput sequencing: Next Generation sequencing
5 points
- Parallel sequencing of millions of short DNA fragments in a liquid or solid matrix.
- Sequencing by synthesis using DNA polymerase.
- Direct or indirect sequence detection via fluorescence
or change in pH. - High throughput allowing sequencing of large genomes
and transcriptomes. - Cost efficiency.
High throughput sequencing: Next Generation sequencing
Steps
- Library preparation for NGS
- Amplification by PCR
(The library is amplified by Polymerase Chain Reaction (PCR) to increase the amount of the template.) - Sequencing
- Sequence assembly
High throughput sequencing: Next Generation sequencing
454 pyrosequencing
3 points
- 4 Enzymes: DNA polymerase, ATP sulfurylase, Luciferase, Apyrase.
- DNA is bound to beads, amplified by Polymerase Chain Reaction and
pyrosequenced in picotitre plates by sequential addition of nucleotides. - High efficiency and low cost: up to 600 million bp sequenced in 10 hours.
High throughput sequencing: Next Generation sequencing
Illumina NGS
4 points
- Parallel sequencing of millions of short DNA fragments on
a solid matrix. - Sequencing by synthesis using DNA polymerase and
fluorescent nucleotides. - High throughput allowing sequencing of large genomes
And transcriptomes. - cost efficent
