Download PCR (BASIC REQUIREMENT, copied from last semester lecture

PCR (BASIC REQUIREMENT, copied from last semester lecture material)
The polymerase chain reaction (PCR) is used for the amplification of short DNA
fragments. PCR differs from PCR in that it is performed in vitro by DNA polymerase and not
in living cells. The PCR technique was developed by Kary Mullis, who was awarded Nobel
Prize in 1993. The following components are needed for the PCR. (1) DNA template
containing the DNA fragment to be amplified. The DNA template can be a complete genome
or a small piece of DNA inserted to a plasmid vector, etc. (2) DNA polymerase executing the
synthesis of new DNA strand. Mullis in his original protocol used an enzyme that degraded
the enzyme at every cycle of the PCR due to the high temperature at the denaturation phase.
This enzyme was later replaced by another DNA polymerase called Taq polymerase, which
was isolated from a bacterium (Thermus aquaticus) living in hot springs. Taq polymerase was
later modified by molecular biological techniques to be optimized for PCR. (3) A pair of
primers (forward and reverse), which are short (18-25 bases) single stranded DNA molecules
with a sequence complementary with the DNA sequence to be amplified. The primers initiate
the synthesis of new DNA strands using the denatured template DNA. We can chose the
endpoints of amplifiable DNA sequence by the selection of primer pairs. (4)
Deoxyribonucleotide triphosphates (dNTPs) ensure the materials and energy for the synthesis
of new DNA strands. (5) The polymerase reaction occurs in the appropriate buffer.
A PCR reaction is composed of 20 to 40 cycles, and each cycle contains three steps. (1)
Denaturation: in high temperature (94-96 ºC) the two DNA strands are separated from each
other (the H-bonds break). (2) Annealing: the temperature is decreased and the primers attach
to the complementary region of template DNA sequences. (3) Synthesis: the DNA polymerase
bind the double stranded DNA (primer + template DNA) and starts DNA synthesis. See video
for the details.
Application of PCR:
(1) PCR can be the alternative of molecular cloning, and it can be combined with molecular
cloning (by ligation of PCR fragment to a plasmid vector
(2) PCR can be used to make mutations in a given DNA sequence.
(3) PCR can also be used for diagnostic purposes, such as detection of genetic and infectious
diseases, as well as for the identification of criminals and for paternity test, etc.
PRIMER DESIGN (Facultative material, will NOT be asked at MTOs or
exams )
Perhaps the most critical parameter for successful PCR is the design of primers. Critical variables are:
 primer length
 melting temperature (Tm)
 specificity
 complementary primer sequences
 G/C content
 3’-end sequence
PRIMER LENGTH
 specificity and the temperature of annealing are at least partly dependent on primer length
 oligonucleotides between 20 and 30 (50) bases are highly sequence specific
 primer length is proportional to annealing efficiency: in general, the longer the
primer, the more inefficient the annealing
 the primers should not be too short as specificity decreases
SPECIFICITY
Primer specificity is at least partly dependent on primer length: there are many more unique 24 base
oligos than there are 15 base pair oligos
Probability that a sequence of length n will occur randomly in a sequence of length m is:
P = (m – n +1) x (¼)n
Example: the mtDNA genome has about 20,000 bases, the probability
of randomly finding sequences of length n is:
n
Pn
5
19.52
10
1.91 x 10-2
15
1.86 x 10-5
COMPLEMENTARY PRIMER SEQUENCES
 primers need to be designed with absolutely no intra-primer homology beyond 3 base
pairs. If a primer has such a region of self-homology, “snap back” can occur
 another related danger is inter-primer homology: partial homology in the middle regions of two
primers can interfere with hybridization.
 If the homology should occur at the 3' end of either primer, primer dimer formation will occur
G/C CONTENT
 ideally a primer should have a near random mix of nucleotides, a 50% GC content
 there should be no PolyG or PolyC stretches that can promote non-specific annealing
3’-END SEQUENCE
 the 3' terminal position in PCR primers is essential for the control of mis-priming
 inclusion of a G or C residue at the 3' end of primers helps to ensure correct binding
(stronger hydrogen bonding of G/C residues)
MELTING TEMPERATURE (TM)
 the relationship between annealing temperature and melting temperature is one of the “Black
Boxes” of PCR
 A GENERAL RULE-OF-THUMB IS TO USE AN ANNEALING TEMPERATURE THAT IS
5°C LOWER THAN THE MELTING TEMPERATURE
 the melting temperatures of oligos are most accurately calculated using nearest neighbor
thermodynamic calculations with the formula: Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log 10
[K+]
(H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the
concentration of primer)
 A GOOD WORKING APPROXIMATION OF TM CAN BE CALCULATED USING THE
WALLACE FORMULA:
TM = 4X (#C+#G) + 2X (#A+#T) °C
 both of the primers should be designed such that they have similar melting temperatures. If
primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the
primer with the higher Tm will mis-prime at lower temperatures; the primer with the lower Tm
may not work at higher temperatures.
Molecular cloning (BASIC REQUIREMENT, copied from last semester
lecture material)
Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining
multiple copies of it in vivo (in living cells, opposed to the amplification of DNA sequences
by PCR).
Restriction enzymes are a group of enzymes that catalyze the cleavage of DNA at specific
sites to produce discrete fragments, used especially in genetic engineering. Also called
restriction endonuclease (RE). REs recognize and cleave a short, typically 4, 6 or 8 base pair
DNA sequence producing 5’ protruding DNA ends (e.g. BamHI or EcoRI REs), 3’ protruding
DNA ends (e.g. KpnI), or blunt DNA ends (e. g SmaI). The restriction recognition sequences
are so called palindromic sequences, which means that reading from the opposite direction (of
the complementary DNA strand also in a 5’→3’ direction) the same sequence is obtained. The
name of a RE refers to the bacterial species and strain from which it was first isolated; e.g.
EcoRI is the first isolate from strain RY13 of Escherichia coli. The natural function of REs is
the defense against bacteriophages. Bacteria defend their DNA against their own REs by
methylation (with methylase enzyme) of their DNA. This is the so called restriction
modification system. Daniel Nathans, Werner Arber and Hamilton Smith were awarded a
Nobel Prize in 1970 for their discovery of REs. Several hundreds of REs are now
commercially available.
DNA ligase can link together two DNA strands that have double-strand break. The
mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’ hydroxyl
ends of one nucleotide with the 5’ phosphate of another. ATP is required for the ligase
reaction. DNA ligases have become an indispensable tool in modern molecular biology
research for generating recombinant DNA sequences. For example, DNA ligases are used
with restriction enzymes to insert DNA fragments, often genes into plasmids.
Plazmid vectors (a) Plasmids A circular, double-stranded unit of DNA that replicates within
a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and
are used in recombinant DNA research to transfer genes between cells. There are many types
of plasmids, for example: (1) Fertility (F) plasmids which contain tra (transfer)-genes. They
are capable of conjugation (transfer of genetic material between bacteria which are touching).
(2) Resistance-(R) plasmids, which contain genes that can build a resistance against
antibiotics or poisons and help bacteria produce pili. (3) Virulence plasmids, which turn the
bacterium into a pathogen. (b) Plasmid vectors Plasmids used in genetic engineering are
called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where
they are commonly used to multiply (make many copies of) or express particular genes. The
gene to be replicated is inserted into copies of a plasmid containing genes that make cells
resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a
short region containing several commonly used restriction sites allowing the easy insertion of
DNA fragments at this location. The replication origo is the site where the DNA polymerase
binds and initiates replication.
Restriction/ligation cloning
In the classical restriction and ligation cloning protocols, cloning of any DNA fragment
essentially involves four steps: DNA fragmentation with restriction endonucleases, ligation of
DNA fragments to a vector, transformation, and screening/selection. Initially, the DNA
fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be
accomplished by restriction enzyme digestion. Following ligation, the ligation product
(recombinant plasmid) is transformed into bacteria for propagation. The bacteria are then
plated on selective agar to select for bacteria that have the plasmid of interest. Individual
colonies are picked and tested for the wanted insert. The bacterial transformation is generally
observed by blue white screening. In this method we utilize the lacZ gene of E.coli lac
operon. For the detection of the lacZ gene product (β-galactosidase) a chromogenic substrate
(X-Gal) is utilized. X-Gal is the substrate of β-galactosidase bit it does not induce the lac
operon. For the induction of lac operon we use another chemical called IPTG, which is not the
substrate of β-galactosidase. Intriguingly, the lacZ gene can be separated to two fragments
(omega: a short fragment at the 5’ end of lacZ gene, and alpha: the rest of the gene) in such a
way that the two peptide subunits bind in the cell and the β-galactosidase retains its
functionality The alpha peptide encoding DNA fragment was inserted to the chromosomal
DNA of E. coli, while the omega subunit was inserted to a plasmid. The omega subunit was
modified to contain several unique restriction recognition sites without destroying its
function. If a foreign DNA sequence is inserted to the omega sequence, we obtain white
plaques, while the bacteria containing the original plasmid vector produce blue plaques in the
presence of X-Gal and IPTG.
RECOMBINATIONAL CLONING:
CLONING WITHOUT RESTRICTION ENZYMES (EXTRA REQUIREMENT)
Although cloning with restriction enzymes and ligase is theoretically easy, in reality sometimes can be very
frustrating. During the recent years, new, recombination based cloning procedures were invented, making
cloning more effective and much faster. One of the recombination cloning method is the so called
GATEWAY system. Instead of restriction enzymes and ligase, we use the integrase enzyme of the lambda
phage. The lambda integrase has different recognition sites. The lambda integrase cuts out any DNA
fragment flanked by attB recognition sites, and inserts it into the place of any DNA fragment flanked by
attP recognition sites. This is the so called BP reaction. The result of the BP reaction is that our gene/DNA
of interest gets incorporated into the donor vector, creating a so called entry clone. In the entry clone our
gene/DNA of interest is flanked by attL sites which originate from the B and P sites upon integrase action.
When we have a recombinant entry clone, we can insert our gene/DNA of interest with a second
recombination reaction to dozens of different destination vectors, with close to 100% efficiency. This is the
so called LR reaction, where the integrase makes recombination between attL and attR sites. The ccdB
gene present in the empty donor and destination vector is used for the selection of recombinant plasmid
containing bacteria, because the protein encoded by this gene is toxic to the bacteria. The bacteria which
were transformed with empty vector or with the byproduct of the LR reaction will die, therefore only the
recombinant plasmid containing bacteria will grow on the petri dish. The empty donor and destination
vector is maintained in mutant bacteria resistant to the effect of the toxic ccdB gene.