WHAT IS DNA? - composition, features, function
DNA is made of two linked strands a double helix. Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups. Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G) or thymine (T). DNA bases pair up with each other, A with T and C with G, to form units called base pairs. It contains all the genetic information needed for an organism to develop, survive and reproduce.
How does DNA-origami work & what can it be used for?
Nanoscale folding of DNA to create 2D/3D shapes at the nanoscale. The specificity of the complementary base pairs is used. Is used for the construction of nanorobots and other structures
How does PCR work and what does it do?
- Denaturation (@94-96°C)
This step is the first regular cycling event and consists of heating the reaction
chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA
melting, or denaturation, of the double-stranded DNA template by breaking the
hydrogen bonds between complementary bases, yielding two single-stranded
DNA molecules. - Annealing (@68°C)
In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F)
for 20–40 seconds, allowing annealing of the primers to each of the single-
stranded DNA templates. Two different primers are typically included in the
reaction mixture: one for each of the two single-stranded complements
containing the target region. During this step, the polymerase binds to the
primer-template hybrid and begins DNA formation. - Elongating (@72°C)
Under optimal conditions ( at each extension/elongation step, the number of
DNA target sequences are doubled. With each successive cycle, the original
template strands plus all newly generated strands become template strands for
the next round of elongation, leading to exponential amplification of
the specific DNA target region. - repeat
-> duplicates DNA
As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute.
How does Agarose Gel Electrophoresis work?
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.
Explain Sanger Sequencing
4 different runs. Each run contains the same DNA different base. Gel electrophoresis is used to separate reads increasing in size in each lane.
The Sanger sequencing method consists of 6 steps:
(1) The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA).
(2) A primer that corresponds to one end of the sequence is attached.
(3) Four polymerase solutions with four types of dNTPs (dNTPs: A, G, C, and T) but only one type of ddNTP are added.
(4) The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated.
(5) The resulting DNA fragments are denatured into ssDNA.
(6) The denatured fragments are separated by gel electrophoresis and the sequence is determined.
Explain Nanopore Sequencing
Enables direct, real-time analysis of long DNA or RNA fragments by driven through a protein nanopore.
It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore, and the molecule bridges the pore. The resulting signal is decoded to provide the specific DNA or RNA sequence, as the amplitude is specific for each of the four bases.
WHAT IS RNA & how it is different to DNA?
Ribonucleic acid. Only one single strand and uracil replace thymine as a complement to adenine. It is essential in various biological roles in coding, decoding, regulation and expression of genes.
WHAT IS Transcription & how does it work?
First, of several steps. Is the process of making an RNA copy of a gene frequency. This copy called messenger RNA leaves the cell nucleus and enters the cytoplasm. Where it directs the synthesis of the protein, which it encodes.
WHAT IS Translation & how does it work?
The next step is after transcription. Here the ribosome is in the cytoplasm or endoplasmatic reticulum and synthesizes proteins. Entier process is called gene expression.
What is southern blotting & northern blotting?
southern blotting & northern blotting are used to determine the identity, size and abundance of specific DNA or RNA(northern) sequences. To do so: DNA/RNA is isolated and gel electrophoresis, then a membrane is transferred to a labelled probe incubation and then target detection.
How many amino acids are used in proteins, and what are their differences? How are they connected?
20! They are classified as essential and non-essentials amino acids. They differ by the structure of the side chain, but all have another atom bonded to the central atom known as the R group.
How are proteins made?
DNA is first transcribed into RNA and then RNA is translated into a protein.
How do enzymes work?
- Enzymes and substrates are in the same area. Some have more than one substrate molecule.
- Substrate binds to a region on the enzyme called the active side.
- Reaction occurs resulting in the new product catalysis happens
- The enzyme releases product.
What is an antibody and where does it come from?
Are Y-shaped proteins used by the immune system to identify and neutralize foreign objects (bacteria, virus) they recognize antigens. They are produced by specialized white blood cells. b CELLS
What is an ELISA and how does it work?
- Antibody coating
- Protein captured
- Detection antibody added
- secondary enzyme linked to an antibody
- Addition of substrate (colourimetric, fluoresence)
- Analysed reflection or fluorescence
How are Antibodies made?
b-cells displaying many different antibodies -> exposure to something foreign. B-cells displaying binders are activated and start to produce soluble antibodies -> antibodies bind foreign “thing” and mark it -> disease is eaten up by macrophages
Explain measurements based on absorbance and Explain measurements based on fluorescence
Absorbance Measurement (colour changes ) / fluorescence ( increased intensity)
Shine light on sample -> light sample interaction -> light detection -> current or voltage measured. Absorbance measures more or less transmission via lambert-Beer A=ECl (linear relationship between the concentration and the absorbance of the solution). For fluorescence, you need an excitation and can then measure fluorescence.
How do immunoassays work, what can you assess with them, give some examples
An immunoassay is a test that relies on biochemistry to measure the presence and/or concentration of an analyte. The analyte can be large proteins, antibodies that a person has produced as a result of an infection or small molecules.
What are microarray-type assays, how do they function, and which types of microarrays do you know of?
Miniaturized high-throughput binding arrays, microarrays provide an easy way to monitor changes in gene expression in the host
There are four different types of DNA microarrays: cDNA microarrays, oligo DNA microarrays, BAC microarrays and SNP microarrays.
What is Flow Cytometry, FACS, and how does it work & does it do? What is the difference towards a simple fluorescence measurement on a plate reader?
Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. FACS is used as a cell sorter and enriched for a subset of cells which is often then studied in further detail using flow cytometry or other analytical techniques.
Explain why there is laminar flow in microchannels.
Because of the small diameter of the channel, the Reynolds number is “always” below 2000 and therefore resulting in laminar flow.
Soft lithography: explain how microfluidic chips based on PDMS are made.
(1) The moulding step allows the mass production of microfluidic chips from a mould. (2) A mixture of PDMS (liquid) and crosslinking agent (to cure it) is poured into the mould and heated at high temperatures. (3) Once it has hardened, it can be taken off the mould.
Which options do you have for making moulds PDMS? If you need chips in large numbers – which methods could you use for production?
PDMS, Photoresist, dry photresist, CNC-milling, reisin DLP
-> large numbers of plastic chips made with injection moulding
What else do you need for using microfluidics apart from a chip? What are necessary peripherals?
Syringe Pump or pressure-driven flow, chip, microscopy/optic solution
