Molecular Analysis of DNA, RNA and Proteins Flashcards
What is a DNA library?
- A genomic DNA library is a set of DNA clones that collectively contain the entire genome of any given organism (eg. E. coli genomic DNA library, mouse DNA library, human DNA library)
What are the two main types of DNA libraries?
- Genomic libraries
- Complementary DNA (cDNA) libraries
What is a cDNA library?
- A cDNA library is a collection of clones containing DNA copies of all the mRNAs expressed in the tissue from which the mRNA was originally prepared
- Therefore, you can have mouse liver cDNA library, or a human intestine cDNA library, or a rat brain cDNA library, etc
Why bother with a cDNA library when you have DNA libraries?
- Only approximately 1/10 of the genome of higher plants and animals is expressed, so it is often better to analyse cDNA when dealing with expressed regions of the genome
- The representation of a particular cDNA clone in a library is proportional to the level of expression of the corresponding mRNA in the original tissue
What is a Poly(A) tail?
- Polyadenylation is the addition of a poly tail to a messenger RNA. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation.
What are the two major parts of constructing a cDNA library?
- mRNA Isolation
- cDNA Synthesis
What are oligo(dT) chains?
- If the mRNA has a poly-A 3’ tail, then an oligo-dT primer can be used to prime all mRNAs simultaneously
What are the first three steps in mRNA Isolation?
- An Elution column contains short oligo(dT) chains linked to cellulose
- The total cellular RNA is isolated from the cells and taken out of the Elution column
- The poly(A) tails of mRNA pair with the oligo(dT) chains and the mRNA is retained in the column
What are the last three steps in mRNA Isolation?
- While the mRNA is being retained in the Elution column, the rest of the RNA is flowing out and being taken out of the column.
- The mRNA is then washed from the column by adding a buffer that breaks the hydrogen bonds between the poly(A) tails and the oligo(T) chains
- This leaves only the mRNA with poly(A) tails
What are the first three steps of cDNA synthesis?
- Oligo(dT) primers anneal to the poly(A) tails of the mRNA and provide 3’-OH groups for DNA synthesis
- Reverse transcription synthesises a DNA strand by using the mRNA as a template
- The RNA-DNA hybrid molecule is briefly treated with RNase, which partly digests the RNA strand
What are the last two steps of cDNA synthesis?
- DNA polymerase synthesises the second DNA strand by using the short, undigested RNA pieces as primers
- The nicks in the sugar-phosphate backbone are sealed by DNA ligase
Study the diagram for Constructing a cDNA library (lecturer mentioned that you may be quized on this)
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What is direct selection?
- A method for the isolation of cDNA encoded by large genomic regions.
How is Direct selection carried out? Give an example.
For example: you want: trpA gene = tryptophan synthase. You have:
- genomic or cDNA library
- Trp- mutant E. coli
- The first step would be to separate the plasmid with the normal trpA gene in it from the mutants in the library
- Step 2: Transform trpA into the E.coli strain (put the plasmid with the normal trpA DNA into the E.coli cells that can’t make tryptophan)
- Step 3: Screen the transformants (put the ones with the recombinant plasmids on a petridish or agar) and let them grow into colonies
Study the diagram for Direct Selection
- google doc
What can be investigated once a gene is cloned?
- Once a gene is cloned its expression, and the function and structure of the protein it encodes can be investigated
- The development of recombinant DNA techniques has spawned new approaches to the analysis of genes and their products
What is Agarose Gel Electrophoresis?
- Agarose gel electrophoresis is a method of gel electrophoresis used in genetics to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar.
What is Agar?
- Agar plates are the standard solid support material for growing microorganisms. Microbial growth media contains nutrients and an energy source to fuel the microbes as they grow, and agar to keep the media in a semi-solid, gel-like state.
What are first two steps in preparing an Agarose Gel Electrophoresis?
- To prepare a semisolid agarose gel with wells for DNA samples you first have to pour the melted agarose into a sealed chamber with the comb in position
- Once the melted agarose has solidified you can remove the comb and sealing tape and then place the gel into the electrophoresis chamber
What are the 3rd and 4th steps in preparing an Agarose Gel Electrophoresis?
- Once the gel is in the Electrophoresis chamber, which contains the buffer, you can load the DNA solutions in the wells of the gel that were made by the comb
- Next you attach the power supply and begin Electrophoresis
What is the last step inpreparing an Agarose Gel Electrophoresis?
- Remove the gel from the chamber after Electrophoresis has finished and stain it with ethidium bromide. You then photograph it under UV illumination
Study the diagrams for Agarose Gel Electophoresis
- Google doc
Who created Southern blotting?
- Ed Southern in 1975
What is Southern blotting?
- A procedure for identifying specific sequences of DNA, in which fragments separated on a gel are transferred directly to a second medium on which assay by hybridization may be carried out
What are the main features of Southern Blotting?
- Allows the identification and locations of genes and other DNA sequences on restriction fragments separated by gel electrophoresis
- The essential feature of this procedure is the transfer of separated DNA fragments onto a membrane (nitrocellulose or nylon)
What are the key steps in performing a Southern blot?
- The DNA of interest is subjected to restriction enzyme digestion to generate smaller DNA fragments
- Digested DNA is separated by agarose gel electrophoresis
- Separated DNA fragments are transferred onto a nylon membrane
- The transfer of DNA is done under denaturing conditions
- Hybridise with labelled DNA prode
What are the first three steps for how the identification of a specific fragment by Southern Blot hybridization is performed?
- A radiolabelled DNA probe containing the sequence of interest is hybridised with the immobilised DNA on the membrane
- The probe will anneal only with DNA molecules on the membrane that contain complementary sequence
- Non-annealed probe is washed off the membrane
What are the last two steps for how the identification of a specific fragment by Southern Blot hybridization is performed?
- The washed membrane is exposed to X-ray film to detect the presence of radioactivity in the bound probe
- After the X-ray is developed, the dark bands show the position of DNA sequences that have hybridised to the probe
What is the difference between Southern Blotting and Northern Blotting?
- Southern Blot: locates a gene from a mixed population of DNA (eg. organisms genomic DNA)
- Northern Blot: locates mRNA that corresponds to gene of interest (eg. used to determine level of gene expression in tissue)
How does Northern Blotting work?
- Similarly to Southern Blotting, first the RNA is separated according to size by electrophoresis through a denaturing agarose gel
- RNA is transferred from the gel to a nitrocellulose membrane
- Prepare a labelled DNA or RNA probe
- The RNA of interest is then located by hybridisation with the probe, which is detected by autoradiography
What is Western Blotting?
- The western blot is a widely used analytical technique in molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract
- It is also known as Immunoblotting
What does Western blotting need to happen during its process?
The western blot technique relies on a combination of the following:
- The resolution/separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
- The specificity of immuno detection using monoclonal or polyclonal antibodies
What is the general procedure of Western blotting?
- Polypeptides/proteins are separated by polyacrylamide gel electrophoresis in the presence of a detergent that denatures the proteins
- Proteins are transferred from the gel to a nitrocellulose membrane
- Individual proteins are detected with antibodies
Why is SDS used in Western blotting and not another detergent?
- SDS is an anionic detergent. Therefore, not only denatures proteins but also impacts an overall negative charge thus allowing proteins to be separate on the basis of size
If SDS is the detergent then wat is the PAGE to make it SDS-PAGE?
- Polypeptides are separated on the basis of size by polyacrylamide gel electrophoresis (PAGE) in the presence of a detergent (SDS) that denatures the proteins
- > SDS-PAGE
How are Individual proteins analysed in Western Blotting?
- Proteins are transferred from the gel to a nitrocellulose membrane
- Individual proteins are detected with antibodies:
- > 1°Ab binds proteins
- > Enzyme conjugated 2°Ab binds 1°Ab
- > Activity of enzyme used for detection
Study the diagrams for Southern Blotting, Northern Blotting, Western Blotting and for the Analysis of Proteins by Western Blot Techniques
- Google doc
