Term 2 Lecture 4: DNA Cloning Continued And PCR Flashcards
PCR process
A way of amplifying a specific piece (region) of DNA many times to produce billions of copies to work with or sequence etc.
Invented by Kary Mullis which earned him the 1993 Nobel prize in chemistry
Process (of one cycle)
1) start with a single piece of dsDNA and heat to 95°C to separate the strands
2) reduce the temperature to 55°C so specific proteins that you’ve added can anneal to the ssDNA
3) raise temperature to 72°C the optimum temperature for a particular DNA polymerase to start copying using the template strand and primer to produce a complementary strand
PCR development 1983-1989
Originally PCR used 3 water baths
1) 95°C add template DNA, primers, nucleotides and buffer - the dsDNA is denatured
2) 55°C primers anneal to template
3)37°C DNA polymerase is added to the mix before it is incubated at 37°C it could not be added before as it would have been denatured
PCR from 1990 to present
Taq polymerase enzyme was obtained from Thermus aquaticus a bacterium in the hot springs of Yellowstone national park
This meant that enzyme did not need to be added for every cycle as it was heat stable so once set up the PCR cycle can be run as many cycles as required
PCR machinery was developed which could cycle through the 3 required temperatures for precise periods of time for the desired number of cycles
Applications of PCR in recombinant DNA technology
1) cloning
a - for screening recombinant clones
b- for adding linkers to DNA fragments prior to cloning
2) gene isolation
a- direct amplification of genes from genomic DNA
b- to degenerate primers for the application of unknown DNA sequences
c- isolation of “tagged” genes from transgenic organisms
3) analysis of gene expression
a- reverse transcriptase PCR (RT-PCR)
b- quantitative or real time PCR (qPCR)
4) DNA sequencing
5) Gene mutation
Other applications for PCR
1) detection of mutations in genes e.g. cancer causing
2) screening for the presence of infectious agents in cells
3) genotyping
4) forensic analysis
Why is PCR so effective?
-exponential rate of amplification due to cycling
- typical PCR reaction is 30 cycles
- number of dsDNA doubles each cycle (2n to n number of cycles)
So a single dsDNA after 30 cycles will be 2³⁰ approx 1x10⁹ (16 billion copies)
Why is PCR so specific?
The primers - there are 4 bases so each has a probability of occuring 1/4
For an RE e.g. Hind lll with a recognition sequence of 6 bases probability of it occuring is 1/4⁶ i.e. 2.44x10-⁴
Therefore in the human genome of 3billion (3x10⁹) bps the number of Hind lll sites would be 7.3x10⁵
The longer the primer/recognition sequence the more specific it is and the less likely it is to occur in a random piece of DNA.
PCR primers are usually DNA oligomers of 18-22 bases therefore probability of an 18mer occuring randomly is 1/4¹⁸ ie 1.44x10-¹¹ - so in the human genome this primer site would occur only 0.04 times - very unlikely.
Also for PCR you need 2 primers so it’s even less likely that both occur randomly, in the right orientation and close enough to produce a product during the extension/synthesis time period (typically 30secs to 1 min)
→ chance of any PCR amplification of any DNA you don’t want becomes infinitesimally small - hence why PCR is so specific
How to design primers
Ideal primers are 19 bases long
Avoid regions of runs of same bases
Ideally 50% GC as they have stronger bonding
Forward primer: binds to non-coding strand so simply select a suitable 19bp section on a 5’3’ strand
Reverse primer: binds to a coding strand so the sequence is the same as a non coding strand - reverse complement of given 5’3’ strand
Primers are simple and cheap to synthesise (about £4 for a 22 base oligonucleotide ordered online from DNA synthesis sites)
Final points for good primer design
Typically 17-22 bases long
Aim for 50% GC
Avoid repeats they lead to mispairing and slippage
Try to avoid primers that can form secondary structures or bind to each other producing short products known as ‘primer dimers’
Screening colonies - colony pcr
Selecting colonies from an x-gal dye plate can be difficult even though a colony appears white it may not have correctly inserted DNA and this can be checked by PCR
Cloning vectors are designed with universal primer binding sites on either side of the multiple cloning sites (MCS)
The primers are commercially available and commonly used for sequencing cloned DNA and amplifying cloned fragments to be run on agarose gel to determine size.
Quick and easy way to check if cloning was successful as usually size of fragment being cloned is known (because primers are designed against gene sequence)
Plasmid doesn’t have to be isolated from a bacteria. A sterile toothpick touched to a white colony and swirled in PCR mix at 95°C bursts cells releasing plasmids and DNA is separated as normal after 30 cycles.
After 30 cycles, loading dye (bromophenol blue in glycol) is added and the sample loaded onto agarose along with a blue colony sample for negative control.
If a band is seen that is the expected size then the bacterial colony that was screened can be grown in nutrient broth and plasmid prep carried out.
Check purified plasmid again by RE digestion then gene clone is ready for downstream application e.g. transferring recombinant plasmid to another organism for gene expression studies.
Possible problems with RE cloning in plasmids
1) may be no suitable RE sites in foreign DNA
2) plasmid sticky ends can reanneal reducing efficiency of cloning reactions
3) fragment needs to be inserted in specific orientation
4) sometimes DNA piece you want to clone is too big for the plasmid
Addressing problems
1) may be no suitable RE sites in foreign DNA: amplify by adding a short extra sequence pre-primer containing an RE site that will be incorporated into the PCR product. RE sites are added at 5’ end of the primer making RE of 6 bases +18-22 fragment
adding RE linker with PCR primer
Before- forward primer - RE region (BAM H1) unattached to the template.
After cycle 1: reverse primer has copied RE site left by forward primer and added Hind lll site (unattached to template)
After cycle 2: Bam Hl and Hind lll sites now are incorporated into SS PCR product
After cycle 3: first ds products with RE sites at both ends are produced
2) plasmid sticky ends can reanneal: reannealing of sticky ends prevented by alkaline phosphate - removes phosphate group either end of the cut site so they cannot reanneal. You don’t treat your fragment so it maintains its phosphate groups so only recombinant plasmids can be produced
3) fragments must be inserted in specific orientation: different restriction enzymes either end so that 2 different sticky ends and therefore DNA can only be inserted in one direction
4) Fragment too big for plasmid : other vectors exist and can be used instead e.g. phage lambda, cosmids or bacterial artificial chromosomes
