Molecular Biology Week 2. Flashcards
Name the five steps involved in DNA isolation.
Release Disrupt and prep cell free extract Remove Debris from extract Purify DNA from extract Measure quantity and quality of DNA.
What is an example of a specimen with nucleated cells?
A common example is WBCs from blood or bone marrow.
Name the long labour intensive method used to separate WBC from the plasma and other components and cells?
Differential Density gradient centrifugation. Ficoll Gradient.
Name the more efficient method of separating WBC from plasma and the other components and cells?
Differential Lysis which uses the osmotic fragility of the cells.
Hypotonic buffer = lysis of RBCs before WBCs. Centrifugation… pellet of WBCs.
What state are tissue samples in before DNA isolation?
Fresh or frozen tissues must be dissociated (separated)
Name the three techniques of dissociating a tissue sample.
Grinding frozen in liquid nitrogen.
Homogenising tissue.
Mincing using a scalpel.
How are fixed embedded tissues deparaffinated?
Through using xylene or less toxic xylene alternatives. Then tissue must be rehydrated.
What can be prepared from the released cells of a tissue sample?
Free cell extract, cells must be lysed.
What do some bacteria and fungi have in common that must be broken down before a cell extract is obtained?
Tough cell walls.
Name the 4 steps involved in obtaining a cell extract from a microorganism sample.
Culture is grown and harvested.
Cells are removed and broken down to produce extract
DNA is purified.
DNA is concentrated.
Name the three conditions which determine cells become a cell free extract.
The cell type (prokaryotic or eukaryotic)
Presence of a cell wall
Composition of the wall.
Name 4 methods involved in preparing cells to become a cell free extract/methods of cell wall and membrane disruption.
Naturally using a gentle buffer.
Enzymes
Grinding or vigorously mixing with beads/magnets
Treatment with detergents and bases such as NaOH
Name three methods of purifying cell free extract to becoming purified DNA.
Organic
Inorganic
Solid Phase Extraction
When purifying DNA which two methods are used in both inorganic and organic techniques. Which one method is unique to inorganic technique?
High salt
Low pH
Inorganic; Mix of chloroform and phenol
What does high salt, low pH and a mix of chloro and phenol do to the contaminants of DNA
Dissolves the hydrophobic contaminants such as lipids and lipoproteins and collects cell debris and strips away most DNA associated proteins.
What is used to degrade RNA?
RNase.
What does chloroform and phenol develop?
Develops a biphasic layer emulsion. After centrifugation; hydrophobic layer at bottom, hydrophilic on top with a white interface in the middle.
What is the white interface in the middle of the biphasic emulsion made up of?
Amphipathic components. Coagulated proteins.
What charge is DNA and RNA and what is its nature towards water?
DNA and RNA is hydrophilic and is negatively charged.
What happens to the test tube with the biphasic layer and the white interface of amphiphathic cells?
Upper hydrophilic layer is collected, DNa is precipitates using ethanol and a high conc of salt.
The Precipitate is collected, rinsed to remove the salt.
DNA is then dissolved in a buffer.
What is the safety issue with phenol?
“salting out” can be dangerous.
How does the inorganic isolation method differ from the organic method in terms of separating the DNA in the solution.
Use of low pH and high salt to selectively precipitate proteins, separating DNA in the solution. Sodium Acetate.
How is DNA collected after being separated in the solution?
It is precipitated in alcohol and then is pelleted and resuspended in buffer.
What is used in solid phase isolation which is different to the two other methods?
DNA extraction is done using solid matrices.
Name the steps of DNA extraction. Solid Phase Extraction.
Sample, lysis (release of nucleic acids) , binding (high salt buffer), washing (buffer), elution (water or buffer) and finally purification of nucleic acids.
How are most solid matrices obtained in a laboratory environment?
Commercial kits in columns of different sizes.
Why is it difficult to work with RNA in the lab?
Due to the widespread nature of RNases. These enzymes can also renature after autoclaving.
Which is the RNA which is most sought after for gene sequencing?
mRNA. Which is 2.5 - 5 % of RNA.
How are the cells lysed when doing an RNA extraction?
This step is done in a detergent or phenol in the presence of high salt or RNase inhibitors. DNase can be added also to remove any contaminating DNA.
What is an example of a detergent which is a strong denaturant in which RNAses can be used?
Guanidine Thiocyanate.
What is the ratio and the chemicals that are used to efficiently extract RNA?
Acid Phenol:chloroform:isoamylalcohol (25:24:1)
How is messenger RNA (mRNA) isolated?
These are required to be isolated in many cases of making mRNA molecules. The polyA tails of the mRNaA molecules are exploited to isolate these molecules.
How are the polyA tails exploited to isolate the mRNA?
PolyT and polyU oligomers bind to the poyA tail founr exclusively on the mRNA. Oligomers of thymine or uracil are immobilised on a matrix of resin column.
What is done after washing away the other RNA molecules?
polyA mRNA is eluted on washing the column with a low salt buffer containing detergent.
How is DNA stored?
As DNa is stable for long periods of time, it can be isolated in a purified DNA (stable at 4C) way or a non-purified (frozen in single use aliquots) DNA way.
What are the three ways in which nucleic acids should be quantified?
- ensuring that extraction was effective.
- ensuring the stndardisation in further analysis of the DNA/RNA
- determining the concentration of nucleic acids relative to the conco of protein that remains in the extracted sample.
Name two methods in which nucleic acids are quantified?
Electrophoresis and spectrophotometric analysis.
What types of applications is gel electrophoresis used for?
To analyse PCR (polymerase chain reaction) products, examine restriction digest patterns etc.
What is Gel Electrophoresis?
It is the movement of charged molecules in an electric field. In which neg molecules move towards the pos electrode and pos molecules head towards the neg electrode.
What are the two types of gel used in molecular biology regarding gel electrophoresis?
Agarose gels and polyacylamide gels.
What is agarose?
It is a polysaccharide that forms gels with pores that vary in diameter depending on the conc of the agarose in the gel.
What determines the range of DNA fragments that can be separated?
The gel concentration. Higher concentration = smaller DNA fragments can be separated.
What are the brief steps to preparing the agarose gel in electrophoresis?
- Mix the agarose powder in a buffer soltion.
- Heat to dissolve, pour molten gel onto perspex plate and tape sides.
- Place comb to form wells for samples.
- Allow to solidify
- 1/2 dyes of known migration rate are added to the DNA before loading.
- Bands of DNA are visualised by using an ethidium bromide/sybr green staining either during or after electrophoresis.
What is ethidium bromide?
This is a fluorescent molecule that intercalates between bases.
How is DNA/RNa analysed using electrophoresis?
It can be analysed by resolving an aliquot of the sample on an agarose gel and generally staining with a fluorescent dye for visual inspection.
The higher the molecular weight genomic DNA = ?
Shows a bright band with low mobility at the top of the gel.
RNA shows up as = ?
two distinct bands of rRNA. quality and ratio
How can DNA be quantiated in gel electrophoresis?
By the comparison of the fluorscent intensity of the sample with controls by v.i and or by densitometry.
At what absorbancy does nucleic acid take in light at through adenine residues?
At 260nm.
What does spectrophotometry do?
It indicates the quality of the nucleic acid. For good quality DNA: 1.6 - 2 x Abs @ 280nm.
Name a development in spectrophometry which allows for fast and reliable DNA and RNA analysis?
Nanodrop technology.
