cDNA Libraries Flashcards
In higher eukaryotic genomes, are coding sequences contiguous or not?
No, genes are organised into exons and introns
What is the size of the haploid human genome in base pairs?
3.24 E9 base pairs
How many protein-coding genes does the haploid human genome encode?
~19,000
What is the size of the average human protein in amino acids?
~500 amino acids
How much DNA encodes the average human protein?
~1500 base pairs
How many base pairs of the human genome is protein-coding?
~2.85 E7 base pairs
What percentage of the human genome is protein-coding?
~0.88%
Where is the protein-coding component found in the human genome?
In the mRNA
What simplifies gene identification?
If regions that align with cDNAs can be identified
What does the cloning of cDNAs help to identify?
Alternative splicing of mRNA
Is it easier to establish a protein sequence from a gene or from a cDNA sequence?
From a cDN sequence
What do we need cDNAs for?
Protein expression
What does transcription of high eukaryotic genes initially result in?
hnRNA, not mRNA
What is hnRNA?
Heterogenous nuclear RNA
How is hnRNA converted to mRNA?
By splicing
What is splicing?
A process by which introns are removed from hnRNA
Give 2 similarities between hnRNA and mRNA
They both have a 5’ cap and a 3’ poly-A tail
What base does transcription usually begin with?
An A or a G
What does guanylyl transferase do?
It adds a guanyl group to the 5’ end in a 5’-5’ bond
What is odd about the nucleases that degrade the 5’ end of mRNA?
They appear to be blocked, due to the cap
How long are eukaryotic mRNA and hnRNA poly(A) tails?
~200 nucleotides
What is the function of the mRNA poly(A) tail?
It stabilises the mRNA
Is the mRNA poly(A) tail encoded in the genome or added as a post-transcriptional modification?
It is added as a post-transcriptional modification
How is the poly(A) tail added to mRNA?
The 3’ end of the transcript is cleaved and a polymerase adds the poly(A) tail
What is the signal for 3’ end cleavage of hnRNA for the addition of a poly(A) tail?
AAUAAA
Genes have just one polyadenylation cleavage signal, true or false?
False, some genes have more than one
What, effectively, is the polyadenylation signal?
The transcription termination signal
What is the outcome of having more than one polyadenylation cleavage signal in a gene?
mRNAs of different lengths
All mRNAs are present in all tissues of an animal, true or false?
False, some mRNAs will be abundant in some tissues and less so or totally absent in others
What would you start with if you wanted to extract all of the RNA from a single animal?
Usually begin with a whole animal (or organ of interest/cultured cells)
Make a series of libraries from each individual organ of an animal
What percentage of total cellular RNA is mRNA?
1-2%
What types of RNA exist in a cell?
mRNA, ribosomal RNA, tRNA, small nuclear RNAs
What method would you use to separate mRNA from other RNAs?
Using the poly(A) tail of mRNA in affinity chromatography
Outline how you would extract mRNA using oligo dT chromatography
- Bind the total RNA (or homogenised cells/tissue) to oligo dT-coated beads in high salt conditions
- Wash away unbound RNA (rRNA, tRNA etc.)
- Elute the mRNA in low salt conditions
Can you clone mRNA directly?
No, so it must be converted into double-stranded DNA
Outline how you would convert mRNA into dsDNA
- Prime first strand synthesis with oligo dT
- Copy the first strand using reverse transcriptase - RNA-dependent DNA polymerase from a retrovirus
- Produces a double-stranded molecule, but one strand is still mRNA
Outline the original method used in the 1970s for converting mRNA into dsDNA
- Synthesise the first strand of cDNA
- Remove mRNA with alkali
- Use the loop to prime second strand
- Remove the loop with S1 nuclease
- Loss of information
Describe the Okayama and Berg method for converting mRNA into dsDNA
Much improved from the original method
Required the production of two vector pieces
Complex procedure for cDNA synthesis
Too complex for most users
Describe the Gubler and Hoffman method published in 1983 for converting mRNA into dsDNA
Developed due to problems with the Okayama and Berg method
Dispensed with the construction of the complex vector components
Retained the RNase H and polymerase I method for making the second strand
Method cited by thousands of authors
How is second strand synthesis of dsDNA carried out in order to replace the original mRNA strand with a DNA copy?
- mRNA is nicked at random points with RNase H
- RNA strand is ‘repaired’ with E. coli DNA polymerase I
- Fragments are joined using T4 DNA ligase
- Ends are ‘polished’ (blunted) with exonuclease
How is cDNA cloned?
The cDNA is ligated into a plasmid vector and then transformed into E. coli
Is blunt-ended ligation efficient or inefficient?
Inefficient
During blunt-ended ligation of cDNA into a plasmid vector, how is the cDNA orientated?
It will be orientated randomly as both ends are blunt
How can the ligation efficiency for cDNA into a plasmid vector be improved?
By adding artificial sticky ends with ‘adaptors’
What do adaptors do?
Introduce sticky ends to both ends of cDNA
During sticky-ended ligation of cDNA into a plasmid vector, how is the cDNA orientated?
Randomly
How can we ensure that cDNA is orientated efficiently in the correct way into the plasmid vector?
- Prime the first strand with oligo dT joined to an adaptor containing an X restriction site
- Ligate Y adaptors to both ends both the double stranded cDNA
- Digest the adapted cDNA with X restriction enzyme
- Ligate directionally into X/Y-digested vector
Making cDNA is very inefficient, what are yields measured in?
Nanograms
cDNA yields are too small to measure so how can cDNA yield be calculated instead?
Use radio-labelled dCTP
A low cDNA yield requires efficient transformation of E. coli and so which transformation methods are preferable?
Electroporation rather than chemical methods
Clones tend to be incomplete with respect to one of the ends of the mRNA, which end is it?
The 5’ end
What does RNase H-deficient reverse transcriptase improve the yield of?
Full-length cDNA
Do most people clone cDNA using kits or manually?
Using kits
What enzymes have improved fidelity and yield of full-length cDNA?
Certain reverse transcriptases (native and engineered)
How would you add a 5’ primer site to cDNA?
- Synthesise first strand of DNA with Mn2+
- Tail the 3’ end of the first strand cDNA
- Ligate a dsDNA adaptor to the cDNA
- PCR amplify using adapter primers or an mRNA-specific primer
What does a cDNA library represent?
All of the mRNAs present in the starting RNA
Will rare mRNAs be rare or common in the cDNA library?
Rare
Why can libraries be normalised?
To reduce cDNAs from abundant mRNAs
Libraries may consist of, say, 1,000,000 clones which are too many to analyse individually, so how can we analyse the clones we want?
‘Screen’ the library
Outline in situ colony screening
- Bacteria are spread on an agar plate and grown
- Colonies are transferred to a nylon filter placed on top of the agar and the plate is kept
- Colonies are lysed onto filter and either the DNA or protein is ‘fixed’ to the filter
- The fixed DNA or protein target is then detected with a specific DNA probe or with antibodies raised against a specific protein
Outline in situ hybridisation
- Grow colonies and blot them onto a filter
- Lyse bacteria and fix the DNA to the filter
- Hybridise a probe to the filter
- Wash away any unbound probe
- Autoradiograph
How many codons to amino acids have?
Most have 4, but some can have 6, 3, 2 or 1
How can you use oligonucleotide probes to screen for a protein in a cDNA library?
- Select a piece of known sequence from the protein whose cDNA you wish to clone
- Choose a hexapeptide with a small number of possible sequence combinations (16 different possible sequences; only 4 uncertainties)
3a. Synthesise oligonucleotides, end label with 32P and hybridise with the colonies
3b. One of the oligonucleotides will have the correct sequence and will hybridise with the colonies containing the desired gene
3c. Some of the pther oligonucleotides may also hybridise to unwanted genes to which they happen to be perfectly matched by chance - Re-screen with a set of probes based on another region of the same protein
What is the assumption when using oligonucleotide probes to screen for a protein in a cDNA library?
That you can purify the protein in sufficient quantity to micro-sequence a bit of it
What types of genomes will oligonucleotide probes work well for?
Small eukaryotic genomes such as yeast but not for a larger genome such as human
What is the size of the yeast genome?
1.2 E7 base pairs
What are oligonucleotide probes based on protein sequence not suitable for?
Screening human genomic libraries
Oligonucleotide probes can be used to screen cDNA libraries which comprise expressed sequences, true or false?
True
Outline how you would screen a library using antibodies
- Purify your protein of interest
- Raise antibodies (polyclonal) by injecting protein into an animal
- Purify antibodies
- ‘Probe’ colonies of library that have been lysed gently to release proteins onto the nylon filter
- cDNA target must be expressed as a ‘fusion protein’
- cDNA is ligated adjacent to a strong promoter, normally into a gene such as lacZ
What does fusion protein expression when screening a library using antibodies require?
The cDNA to be cloned into an expression vector
What is absolutely required for a fusion protein to be expressed in an expression vector?
The fusion protein must be in the correct orientation and reading frame: use directional libraries, but still only 1 in 3 genuine cDNA clones will react with the antibody
How can you confirm the identity and correctness of a cDNA clone?
By sequencing it
DNA sequencing was developed simultaneously by which two groups in the late 1970s?
Maxam and Gilbert using chemical cleavage
Sanger using dideoxy chain termination
