Lecture 18 - 24 Flashcards
In the procedure of making a genomic library and cDNA library, what is the key difference?
cDNA library isolates the mRNA in a cell rather than the DNA. The mRNA then goes through reverse transcriptase to become cDNA. The cDNA then has restriction sites added to the ends of it and the procedure from there is the same as genomic library.
How is a genomic library made (directional cloning)
The cell of interests DNA is isolated (with restriction endonuclease) and at the same time digest a plasmid vector (with a MCS) with a single endonuclease. Treat the plasmid DNA with phosphatase to remove 5- phosphate groups. Then mix the isolated DNA and plasmid with t4 DNA ligase to join them together. Transform the DNA and then plate on agar
What is the transformation method
Prepare competent cells, COME BACK TO THIS
List ‘Key Rules’ for PCR primer design
- At least 17 base pairs, usually 40-60% GC
- Avoid complementary sequences (avoid hairpins)
- Forward and reverse primers should have similar Tm.
What factors need to be considered when selecting tissue to make a cDNA library?
Expression level will differ in different sites
Accessibiliy of tissue
Select tissue at appropriate stage of development
The RNase level within tissue
List differences of a genomic library compared to a cDNA library
Genomic: gene coding sequence is fragmented
- Recombinant plasmid may contain sequence
info from more than one gene
- Contains introns
- contains promoter and regulatory
sequences
- Preparation of library involves restriction
digest of source nucleic acid
- sequence of interest represents 0.001% of
total library
Similarities between genomic and cDNA libraries
Libraries include 5’ and 3’ untranslated regions of gene sequences
Preparation of libraries uses restriction endonucleases and ligase
What is the difference between non-directional and directional cloning?
Directional cloning - plasmid and insert DNA is cut with two restriction enzymes.
Non-directional cloning - plasmid is dephosphorylated to prevent self ligation
Directional cloning - recombinant plasmid only has one orientation.
What are the steps of Directional cloning
- Isolate and cut plasmid with restriction enzyme
- Dephosphorylate and purify vector fragment.
- Cut insert DNA with restriction enzyme and isolate
- Combine insert DNA with cut plasmid
- Add DNA ligase to form covalent bonds
- Introduce plasmid to bacterium (transformation)
- Plate bacteria
- Culture individual clones
- Harvest and analyse recombinant plasmids
- Express and harvest recombinant protein.
List ways to identify recombinant plasmids
- DNA sequencing
- PCR using combination of vector specific and insert - specific primers
- Replica plate using different antibiotic
- Grow cultures, harvest plasmids, restriciton digest using enzyme that cuts vector and once off-centre in insert. Analyse results using gel electrophoresis
- Induce expression, look for protein expression on a protein gel.
What is Klenow
Klenow is a DNA modifying enzyme that can be used to make blunt ends by removal of 3’ extension or filling in 3’ recessed end
What does alkaline phosphatase do?
removes the 5’ phosphate groups to prevent cut plasmid from ligating with itself.
What is required for T4 DNA ligase and E.coli ligase and what ends can they join?
T4 DNA ligase requires ATP and can join both sticky and blunt ends
E.coli ligase requires NAD+ and can only join sticky ends.
In blue/white screening, what does Amp, X-gal and IPTG do?
Amp - selects for recombinant plasmids
X-gal - has its glycosidic bonds cleaved and turns cells blue (negative result)
IPTG - Inducer for lacz(alpha) peptide
What is alpha-complementation
Alpha complementation is when Lacz(alpha) encoded by empty plasmid associates with Lacz(omega) encoded by the host to produce beta-galactosidase.
In blue/white screening, why are recombinant clones white? What is insertional inactivation?
The insertion of a foreign DNA fragment into the multiple cloning site inactivates the lac(alpha) gene thereby prevents the formation of beta-galactosidase (example of insertional inactivation). beta-galactosidase is required to cleave the glycosidic bonds in X-gal which turns the clone blue.
What are situations where blue/white screening give false negative results? (blue colonies that have foreign DNA)
Insertion of a small fragment that lacks a stop codon.
Use of incorrect cloning strain that doesnt support blue/white screening
What are situations where blue/white screening give false positive results? (white colonies that don’t have foreign DNA)
Mutation of lacZ(alpha) or promoter on plasmid
Loss of expression of lacZ(omega) fragment of beta-galactosidase.
In identifying recombinant clones, what is replica plating?
COME BACK TO
What is insertional inactivation?
Insertional inactiviation is insertion of a foreign piece of DNA that inactivates ability to produce a gene.
List the requirements for Sanger Dideoxy sequencing method
- Single stranded DNA template to be sequenced
- Short oligonucleotide primer to provide free 3’ OH
- DNA polymerase to catalyse chain elongation
- dNTPS (dATP, dCTP, dGTP, DTTP) act as substrates
- radioactive labelled dNTP or primer to visualise
- dideoxynucleotide to block further elongation
In constructing a cDNA library, how is mRNA turned into cDNA?
- Oligo dT primers are added which anneal to the polyA
tail of mRNA and provide 3’ OH groups for DNA
synthesis - With the help of dNTPs, reverse transcriptase
synthesises a DNA strand using mRNA as template - RNase H is added to patrially degrade the mRNA
- DNA pol I is added to synthesise second strand of
cDNA using mRNA remnants as primers - DNA ligase then seals phosphate backbone up to
make double stranded cDNA
Advantages of cDNA library compared to genomic library
- cDNA lacks introns so ORF can be easily read
- cDNA clones are intact
- Sets of cDNA derived from a single gene may differ
- Can select tissue in which a particular gene is
expressed highly. - Sequence of interest can represent 40% of total
library
How is cDNA inserted into a vector
DNA ligase combine cDNA and linkers to end of cDNA
What is a probe and how do you obtain probes
Probes are single stranded labelled nucleic acid with sequence matching the gene of interest. They are obtained through PCR, cloned DNA or synthetic oligonucleotide
How are probes designed
- Select amino acid sequence with minimal degeneracy
- Determine all possible nucleotide sequences that
could encode that amino acid sequence (use product
rule) - order a set of degenerate oligonucleotides matching
those DNA sequences
How to screen a genomic library with a radioactive probe
- Plate colonies from library
- Overlay membrane filter on plate
- Denature DNA (turn single stranded) on membrane
Akali - Add radioactively labelled probe
- Wash off unbound probe
- Expose X-ray film
- Develop film
- Compare film with original plate
