Molecular Lab Techniques Flashcards
Four major classes of biological molecules that play essential roles in all organisms.
- Nucleotides
- Amino acids
- Carbohydrates
- Lipids
Model organism
A model organism is a non-human animal or plant species that is conveniently studied to understand particular biological phenomena , with the expectation that discoveries made in the model organism will provide insight into the working of an organism of interest.
Most commonly use model organisms
- Fruit fly ( Drosophila melanogaster)
- Hermaphrodite worm (Caenorhabditis elegans)
- Arabidopsis thaliana plant
- Mouse model (Mus musculus)
When do you use centrifugation and cell fractionation.
When you would like to isolate different organelles, or separating soluble and membrane-associated molecules from other cellular components.
What are the scientific principles of the centrifugation and cell fractionation.
- Intact cells must be disrupted to release their contents.
- Individual components can be separated by the centrifuge.
What methods can be used to release cell contents from a cell.
- Ultrasonic waves
- High pressure
- Detergents that generate holes in the cell membrane
How does the centrifuge work?
The centripedal forces that results when samples are accelerated at upwards of 100000g causes large molecules to move toward the bottom of the tube and form a pellet.
Differential centrifugation.
Repeated centrifugation at progressively higher speeds will fractionate cell homogenates into their components.
How Gel electropheresis works.
The negative charge of DNA and RNA molecules are used too seperate the DNA and RNA molecules of different sizes. This method relies on a porous gel made from agarose or polyacrylamide through which nucleic acids migrate when the gel slab is immersed in buffer and an electrical field applied.
How do we make sure proteins seperate in gel electrophoresis in a correct way?
The proteins must first be denatured and then coated with a negatively charged detergent.
What are Blotting techniques used for?
They are used by scientists to detect specific RNA,DNA or protein species in a sample.
What are subtypes of blotting techniques such as?
- Southern blotting
- Northern blotting
- Western blotting
- South-western blotting
- Eastern blotting
Why are seperated molecules transferred to a membrane after gel electrophoresis.
Proteins and nucleic acids have difficulty traveling through a gel. Once a protein or nucleic acid sample has been separated, you will need to readily access the separated molecules for detection with a nucleic acid probe or antibody. By transferring the molecules to a solid, sticky surface, they can easily be exposed to the probe or antibody you are using to detect a specific sequence or protein.
Sanger sequencing
- Production of chain-terminated DNA fragments from a common template, where each terminated chain incorporated a dideoxynucleotides (ddNTP) with a specific fluorescent label.
Dideoxynucleotides
Dideoxynucleotides or ddNTPs are nucleotides lacking a 3’-hydroxyl (OH) group on their deoxyribose sugar. This results in termination of elongation because the dideoxynucleotides cannot bind another dNTP.
What is gene cloning ?
Gene cloning involves taking a piece of DNA from the organism where it naturally occurs and putting it into a host cell such as the bacterium.
Why is it important to have selectable markers on cloning vectors.
Because only about one in a million of the bacterial cells successfully take up the plasmid.
When do you use recombinant protein expression.
You use it when you wish to have a physical sample of a pure protein of interest for an assay.
Steps of recombination protein expression.
- PCR products are inserted expression vectors.
- Recombinant vectors are inserted into E.coli.
- Recombinant E.coli are planted on antibiotic-enriched media and DNA is sequenced into a protein in the recombinant E.coli.
- Protein is extracted, it can be run on an SDS-PAGE gel to be visualized.
When do I use PCR.
- To detect the precence of a DNA sequence in a sample.
- Amplify and Isolate a gene for cloning
- Amplifying a gene so that it can be sequenced and studied.
- RT-qPCR is used when you want to quantify the expression of a gene at RNA level.
Southern blotting steps
- Nucleic acids seperated according to size by electrophoresis in an agrosegel.
- Separated nucleic acids blotted onto membrane by suction of buffer through both gel and paper.
- Membrane with seperated nucleic acids hybridized with labeled probe.
- After removal of unbound probe, bands complementary to labeled probe are revealed by specific detection of label.
What questions do nuclear run-on assay answer.
- Is a particular gene regulated primarily at the level of transcriptional initiation, or regulated by downstream events.
- How does the rate of transcriptional activation/ initiation compare under different experimental conditions, or compare between different genes.
Advantage over nuclear run on assay
- Resulting are not influenced by RNA degration, since degradation takes place in the cytoplasm, but radioactive labelling is preformed within the isolated nuclei.
- By removing cytoplasm, the large pool of unlabelled NPTs is removed, therefore more labelled.
When and why would i perform RT-qPCR.
When we want to compare levels of one or more transcripts between samples with high accuracy and high sensitivity. It can detect very low concentration of cDNA that northern Blots or DNA microarrays cannot.
Quantitative PCR
By adding a fluorescent dye to the reaction that intercalates into the dsDNA product, a specialized thermocycler can measure the amount of PCR product present at every cycle using a laser and detector.
What does RT-qPCR help us acchieve.
Tracking amplification in real time with the fluorescent dye allows us to identify which samples start to reach exponential amplification first which indicates which sample had the most cDNA(the gene is highly transcribed)
How do Northern blots work.
A DNA probe is used to detect RNA.
How does western blot work
The use of an antibody directed against a protein of interest.
What are the steps involved in next generation DNA sequencing.
- DNA extraction
- DNA fragmentation
- High-throughput sequencing
- Bioinformatic analysis
What does the amount of mRNA bound to each site in microarrays mean.
It indicated the expression levels of the various genes.
What questions can i answer with 2D-page
This technique is used when you want to identify novel proteins that are expressed between two sample. It is useful when you have no prior information on proteins that differ between samples, and no antibody to detect them with.
The steps of principle of 2D-page.
- Seperated by charge with isoelectric focusing, which seperates proteins via pH.
- The seperated protein on the gel with IEF is negatively charged by treatment with SDS, and the electrophoresis is performed.
What questions can be answered by doing a DNASE1.
- How is the chromatin packaged at a particular locus.
- What are the physical position of nucleosomes in the genome or at a particular locus.
What are the basic steps of DNASE 1 and micrococcal nuclease assay.
- The cell is lysed
- The nuclei is extracted
- The nuclie is made permeable
4 the chromatin is digested with DNase 1 or MNase at 37 degrees celcius . - Digested DNA is then extracted and quantified with qPCR.or next gen sequencing.
How does DNASE 1 and micrococcal nuclease assay work.
DNase is partially inhibited by all DNA bound proteins and will cleave the most exposed DNA first, this allows us to see which DNA in the chromatin is the most exposed.
Whatt question can i answer with bisulfide sequencing.
This is a very specific and powerful assay to find out which cytosine are methylated in a specific dna sequence, in a specific sample relative to another .DNA methylation is dynamic and highly variable across tissue, cell lines, treatment and time periods.
How does Bisulfite sequencing.
Bisulfite causes de-amination of unmethylated cytosine and converts them into uracil. After PCR amplification, the resulting Us are replaced Ts. Methylated Cs are immune to conversion by bisulfite and remain as Cs.
Basic steps of Bisulfite
- Isolate gDNA and divide each sample into two. The gDNA will retain the methylation found in vivo.
- For one of each duplicate sample, and bisulfite. The other is the unconverted control.
- PCR-amplify the target sequence of interest using gene-specific primers for both the bisulfite-treated and unconverted control.
- Sequence the PCR fragment for both the bisulfite-treated and unconverted control.
What does Chromatin immunoprecipitation.
Chromatin immunoprecipitation Assays can specifically determine the binding sites of modified proteins apart from unmodified versions. We can also use it to identify methylated DNA, but not a single-nucleotide resolution.
What does dnase 1 footprinting assay accomplish.
Helps us figure out which specific positions in a promoter are bound by proteins.
How does DNASE 1 Footprinting
- DNase 1 is more likely to cleave naked DNA than DNA bound by a protein. When digesting a piece of DNA, some of which is occupied by transcription Factors (TFs), the DNase 1 will first nick naked dsDNA found in between the protein.
- The nicks results in fragments of many different lengths, they can be detected with a blot and any gaps in the size range indications positions on the promoter Where proteins were bound.
What research Questions does electrophoretic mobility shift assay.
- Does a protein in a particular cell type bind to the sequence of a promoter region.
- To which nucleotide sequences does the protein bind.
- How specific is this binding to the DNA sequence
- Does a known protein bind to this sequence
The specificity of the protein of the DNA sequence is investigated by competition experiments by using:
- Non-specific DNA competitor
- Radioactively labelled specific competitor
- Not labelled specific competitor
What are reporter genes
Reporter genes encode a product that can easily be detected and quantified.
Examples of reporter genes.
- CAT
- Bacterial LacZ gene
- Luciferase
- GFP
THe CAT reporter genes
Detect radiolablled product by autoradiography.
Bacterial LacZ gene reporter genes
Blue product when provide with X-gal substrate
Luciferase reporter gene
Fluorescent protein , detected with fluorometer
GFP reporter genes
Fluorescent protein encoded by gene of a jelly fish, one of the most popular reporters, versatile and stable , fluoresces bright green when exposed to UV light, no substrate needed.
How do we investigate if chromatin structure plays a role in gene regulation.
- Use endonuclease that cleaves DNA.
- MNase assay
.3. DNase 1 sensitivity assay - DNase 1 hypersensitivity assay
What can detect methylation status of Cs in entire genomes.
Sodium bisulfite assays
The principle of sodium bisulfite assay
When DNA is treated with sodium bisulfite, it will convert unmethylated Cs to uracil but have no effect on methylated C’s
Principle of chromatin immunoprecipitation (ChIP)
Antibodies that specifically recognise and bind to methylated Cs are used to immuno-precipitate chromatin containing methylated C’s
What is deamination.
Removal of NH2
What does deamination convert
- Methylated cytosine is converted into thymines. (Thymines are retained)
- Unmethylated cytosines are converted into uracils ( repaired back to unmethylated cytosines)
What catalyse de novo methylation of unmethylated C’s.
Dnmt3a and Dnmt3b
How can methylation of specific sites on DNA be lost .
- Active demethylation, catalysed by DNA demethylation enzymes.
- Passive demethylation, catalysed by inhibiting the action of the maintenance methylase.
