Topic 4 - Recombinant DNA technology 1 Flashcards
Cloning DNA involves what?
producing copies of DNA fragments
What are the tools of recombinant DNA technology…?
- Restriction endonucleases (enzymes)
- Agarose gel electrophoresis
- Plasmids
- DNA ligase
- E. coli
What are restriction endonucleases functions…?
- enzymes isolated from BACTERIA cut at specific sequences (4-8 base pairs)
- protect bacteria from foreign DNA (beef, lamb, plant material…) by cutting them before they get into cell
How do cells protect their own (bacterias) DNA from ‘cutting’ by restriction endonucleases?
bacteria protect DNA by modifying it by adding methyl groups (-CH3) so restriction endonucleases can’t latch on and cut DNA
What is EcoR1…?
Restriction enzyme that cuts DNA sequence (GAATTC) -> ‘sticky 5’ end’
R - strain designation
1 - represents 1st enzyme
Restriction endonucleases (RE’s) can leave which kinds of ‘ends’ after cutting…?
- blunt ends (Rsa1) -> no overhang
- Sticky 5’ ends (EcoR1) -> 5’ overhang
- Sticky 3’ ends (Kpn1) -> 3’ overhang
How is the frequency of RE sites calculated?
…by the number of base pairs
eg. 6bp sequence = 4^6 = 1 every 4096 bp’s on average
Therefore a shorter sequence will be found much more often than longer sequence
Cleavage cites are …?
palindromic (RADAR) or (GAATTC)
A bit about electrophoresis…
- Electrical field applied over an agarose or polyacrylamide gel
- Agarose is a carb polymer from seaweed
- pores in agarose act as MOLECULAR SIEVE
- DNA is -vely charged (phosphate group)
- Mobility depends on size AND conformation
Joining DNA fragments is called …? What does this?
Ligation by DNA ligase ‘molecular glue’
How does DNA ligase work? What is required for ligation?
…reforms phosphodiester bonds (S-P) joining fragments together.
Requires: ATP & magnesium
DNA ligase is isolated from …?
bacteriophage T4
A bit about plasmids…
- small ~2000 - 20,000bp
- circular pieces of DNA found in bacteria (mostly circular, yeast plasmids can be linear)
- carry genes for AB resistance why?
What is conjugation?
the act of passing plasmid from one bacterial cell to another (bacteria sex)
Why do plasmids carry genes for antibiotic resistance?
To allow them to survive in hostile environments. bacteria compete with fungi for nutrients & fungi produce AB (eg. penicillin) They do this by secreting AB resistant proteins into medium around them -> degrade ABs
What are plasmid vectors?
natural plasmids that have been genetically modified to make them more useful for research etc. ie. transferring DNA into bacteria
Useful characteristics of plasmids for cloning DNA…?
- selectable markers (AB-resistance genes)
- single restriction enzyme sites (for inserting DNA)
Usual bacterial host for plasmids?
Escherichia coli (E. coli)
What is a ‘single restriction enzyme site’?
site for DNA insertion into plasmid
One of the first plasmids used in genetic manipulation?
Plasmid pBR322
Which AB resistance genes does pBR322 contain? What are the 3 single sites for restriction enzyme?
AB resistance genes - Ampicillin - Tetracycline Single restriction enzyme sites - Pstl1 - EcoR1 - Sal1 Origin of replication
What are the 4 stages of inserting DNA fragment into a plasmid…?
- CUT/isolate DNA from source (donor DNA), cut plasmid vector with same enzyme from culture of E. coli
- LIGATE DNA using DNA ligase to join DNA fragments together after mixing the 2 DNA fragments
- TRANSFORMATION or transfer into E. coli
- SELECTION of E. coli containing recombinant plasmids
What must all plasmids contain?
origin of replication (to be able to replicate in the cell)
What is transformation?
uptake of DNA by a cell (E. coli)
Cultures are treated with chemicals such as …?
calcium chloride (CaCl2) - commolnly used heavy metals
When cells become porous to take up DNA, they are said to be …?
‘competent’
Re. transformation, cells take up plasmids at … frequency?
…at very low frequency (1 cells in thousands) - very inefficient which is why cells are given ‘heat shock’ ~42 degrees for 90sec -> Lbroth medium for growth
What are ‘selectable markers’? Eg’s of markers..?
Variety of AB resistance genes to select bacteria
eg. ampicillin, tetracycline, kanamycin
When recombinant plasmids are added to population of E. coli cells then plated onto agar medium containing ampicillin, which cells will grow?
only cells containing recombinant plasmids (ampicillin resistance gene)
How can you make sure that recombinant plasmids are present as opposed to regular re-circularised ligated plasmids?
Insertional inactivation:
Plasmids frequently designed with multiple cloning sites (MCS) - short region of DNA within plasmid with many sites for single restriction enzymes
Where is the multiple cloning sites (MCS) located?
Within lacZ gene on plasmid
If a colony on agar is blue coloured, it has …?
bacterial colonies with plasmids containing beta-galactisidase
beta galactosidase + X-gal = ?
blue coloured compound
What colour are colonies with foreign DNA present in the MCS? Why?
white, because beta-galactosidase gene gets disrupted by its presence thus inhibiting its usual blue colour
What are genomic libraries? What do they require?
collection of DNA fragments representative of entire genome of an organism
Require DNA fragments (cleaved by restriction enzyme), insertion into plasmids (via DNA ligase), bacteria as host (E.coli)
Why/when are genomic libraries useful?
can be used to select certain genes eg. gene for human insulin can be looked for in a human genome library or if a particular sequence in a genome or gene is desired, can use genomic libraries as resource for that
What are the different vectors…? Smallest to largest DNA holding capacity
plasmids, bacteriophage lambda, cosmid, bacteriophage P1, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC)
Properties of plasmid vectors…?
double-stranded circular DNA
host: E. coli
up to 15k bp used in cDNA libraries & subcloning
Properties of bacteriophage lambda vectors…?
virus linear DNA
host: E. coli
up to 25k bp used in genomic & cDNA libraries
Properties of cosmid vectors…?
double-stranded circular DNA
host: E. coli
30-45k bp used in genomic libraries
Properties of bacteriophage P1 vectors…?
virus circular DNA
host: E. coli
70-90k bp used in genomic libraries
Properties of bacterial artificial chromosome (BAC) vectors…?
BAC
host: E. coli
100-300k bp used in genomic libraries
Properties of yeast artificial chromosome (YAC) vectors…?
YAC
host: yeast
250-2000k bp used in genomic libraries
Human genom is … bp long?
3 billion bp ~ 2.9 x 10^6k bp (2.9 x 10^9 bp)
What is the equation to work out minimum number of clones required with a 95% probability of having at least one clone in the library?
N = ln(1-P)/ln(1-a/b) where, P = probability (0.95) a = mean size of fragments b = size of genome ln = natural logarithm
What % of human genome is non-coding? ie. does not code for protein
90%
Why would we make complimentary DNA?
making library of genes expressed & not genes that aren’t, we don’t want to clone unneccessary DNA as 90% of human genome is non-coding
What does complimentary DNA do in eukaryotic genes?
in eukaryotic genes containing introns (non-coding sequences) so making copies of mRNA -> DNA form is useful as they have already had their introns removed
How do we obtain complimentary DNA library?
isolate mRNA (reverse transcriptase) -> DNA -> clone DNA
Where does reverse transcriptase come from?
avian retrovirus (RNA viruses that can infect cells & insert their RNA genome in DNA form into the chromosome)
Process of making cDNA from mRNA of RBC…
extract RNA from RBC -> RBC mRNA (poly A tails), add synthetic primer (oligodT primer) single stranded T nucleotides bind to poly A tails -> cDNA & mRNA hybrids -> gets to end and curls over with hairpin loop structure and stops as no RNA template. Add normal DNA polymerase (DNA-dependent DNA polymerase) uses cDNA as template for synthesis. mRNA strand get digested by treating solution with RNAse. To get rid of hairpin loop use single-stranded nuclease (S1 nuclease) to cut hairpin loop end is single stranded -> cDNA double helix -> insert into plasmid vector
What are cDNA libraries useful for?
To isolate particular genes of interest, and also can be sequenced, also identify which parts of genome encode protein-coding genes
Look at diagram of cDNA on slide 31
maybe even youtube…
Strategy for cloning cDNA for insulin gene…?
pancreas -> isolate mRNA (proinsulin) -> reverse transcriptase -> cDNA (plus other genes) -> into plasmids (recombinant plasmids) -> infect E. coli = insulin produced in the bacteria
Is any cDNA for any gene in human genome transferred into plasmid vector in E. coli likely to produce functional protein? Why?
No, firstly because of the presence of introns in many genes, and secondly because you’d need a bacterial promoter for that gene to be expressed
What do genomic libraries allow us to study?
gene structure - promoters to know how gene is regulated or entire sequence of genome
What do cDNA libraries allow us to do?
express genes in other organisms eg. insulin or arabinose promoter
T or F - libraries can be stored indefinitely
true - by freezing
1000’s of clones in library, but want to find a particular gene encoding desired protein of interest eg. insulin. How do we screen libraries for particular genes?
antibody screening
How does antibody screening work? eg. for insulin gene
take plate with colonies & overlay it with moist NITROCELLULOSE paper to produce imprint of plate -> absorbs cells onto paper -> treat with NaOH -> release proteins which adhere to paper -> incubate paper in buffer of labeled insulin Ab -> take Ab for insulin gene -> binds to insulin protein -> wash excess Ab’s off & illuminate paper with UV light -> colony binding Ab lights up -> transfer paper back to original agar plate and compare to identify locations of colonies of insulin gene -> purify to see whether they produce insulin
Basically it can screen many (100’s of 1000’s of colonies -> 1 or 2 colonies containing gene of interest)
Screening by hybridisation? When would this process be used? Why can’t you use an Ab to screen for this?
When searching for insulin gene in genomic library or cDNA in non-expression library.
Cannot use Ab because likely that DNA is not going to be expressed thus proteins won’t be produced
How does screening hybridisation work?
heat or chemically treat DNA fragments -> destroy H-bonding in double helix -> single stranded -> cool fragments then complimentary strands will find each other & base pair
How does screening hybridisation work? In full detail…
Non-expression cDNA library in agar plate -> overlay nitrocellulose paper -> peel it off & treat with NaOH which lyses the cells & denatures DNA -> bake or treat with UV so DNA binds to plate/paper. If looking for beta globin gene, take gene and label with RADIOACTIVE ISOTOPE (32p) & heat DNA to denature -> single stranded -> incubate in suitable buffer -> cDNA finds complimentary strand & stick to it at around 60 degrees -> wash excess DNA from filter & expose to x-ray film -> image with colonies on agar plate & dark colony will indicate colony exposed to radioactive activity and labeled DNA with beta globin genomic DNA clone
Must characterise a clone first!
What percent complimentarity must be obtained between probe (cDNA) & its complimentary sequence in colony?
80% nucleotides need to be same or better
What is a DNA probe?
labeled piece of DNA that can be used to find another piece of DNA by hybridisation
What can DNA consist of…?
cloned (cDNA)
genomic DNA
synthetic oligonucleotides
Does complimentarity need to be 100% in DNA probes?
No, eg. human insulin gene can be used to find a horse homologue
