pcr

it is mainly controlled by use of thermal cycling

thermal cyclers make use of peltier effect.
Sample dna is denatured at 94 degree
Primers are added at 54 degree then temperature is raised to 72
degree and annealing takes place (why do we need to increase the
temperature? Is it because of taq polymerase)
Extension of strands also depends upon the temperature modulation.

Components for pcr

Dna template containing dna region

2 primers that are complementary to 3-end of each dna
Forward primers indicates the START of PCR. Its sequence is the
same as 5-3 template DNA sequence. How?
Reverse primer indicates the END of the PCR. Its sequence is the
reverse complement of template DNA.
PCR Primers are short single-stranded, synthetically synthesized
oligonucleotides 18-25 nucleotides.
Melting temperature of the primers is the most important
characteristic. Ideally between 54-60C and as similar as possible to
each other. Why? (Then why is that for extension temperature is raised
if it exceeds melting point of primers?).
If the annealing temperature is too low then non-specific annealing will
occur.
For extension temperature is raised because taq polymerase works
well at 72 degree.
For primers containing less than 25 nucleotides, the approx. melting
temperature (Tm) can be calculated using the following equation:
Tm = 4 (G + C) + 2 (A + T), where G, C, A, T number of respective
nucleotides in the primer
Rules for primer design.
o Base composition should be 40-60% (G+C); ideally C and G
should be distributed uniformly.
o primers should end (3') in a G or C, or CG or GC; - runs of three
or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences and should be avoided; Why?
o Check for possible additional complementary sites between
primers and template DNA
o Tms between 55-80C are preferred; Tm for two primers shall
not differ by more than 5C.

Taq polymerase
How can you get pcr product with use of regular dna polymerase?
Dntp (nucleotides)
Each dNTPs should be present in equimolar concentration.
The concentration of dNTPs in PCR reaction should be around 50200M.
If the concentration of dNTPs is high the fidelity of the process will be
adversely affected by driving Taq DNA polymerase to misincorporate
at a higher rate than normal
While if the concentration is lower it may affect efficiency of PCR.
Buffer solution for providing optimum activity and stability of dna
polymerase.
The general composition of 10X buffer: -100 mM Tris H-VI (pH 8.3 at
250C ) 500 mM KCl+15mM MgCl2 +1mg/ml gelatin+0.1% tween
20+0.1% NP40
Divalent cations are used for mutagenesis (eg. Mg2+ , Mn2+)
Exists as dNTP-Mg2+ complexes,
Interacts with DNA backbone ( what it actually does by interacting
with backbone?)
Essential cofactor for Taq polymerase
The total magnesium ion concentration must exceed the total dNTP
concentration, for it to act as cofactor.
Low concentrations tend to have low yields of PCR product.
High concentrations tend to reduce the fidelity of Taq polymerase and
lead to amplification of non-specific products.
Monovalent cation- potassium ions.
Other Components
The salt used in most reactions is K / Na, added to facilitate correct
primer annealing.
Other components stabilize the enzyme are: gelatin, bovine serum
albumin or nonionic detergent(Tween 20).

Lecture 12-13
Cloning methods

A High-throughput cloning system shall have:

Fidelity: cut and paste type to avoid mutagenesis.
Ease of use: Minimum number of steps, tolerance to variables.
Reliability: Well optimized kit format
Flexibility: One master clone for all applications

 Undesirable sequence shall be at minimum

Low cost
Expression cloning system? (what is n-terminal and c-terminal and there
difference?)
RE based classic method of dna cloning is not effective for high-throughput
cloning because the commonly used REs have recognition sequences that
occur too frequently within ORFs. The exception to this is a RE-based method
called Flexi cloning system from Promega.
Flexi cloning system
Q. how does gene from other flexi vector acts as barnase lethal gene when
transformed in other vector?
Other interesting information
N-terminal tagged protein configuration: (tag SgfI GOI PmeI)
Protein expression starts from inbuilt AUG start codon of tag
Ends with stop codon of GOI or at PmeI.
SgfI adds only 3 amino-acids: Ala-Ile-Ala.
SgfI RE site:
GCG ATC GC
CGCTA G CG
Ala Ile Ala
PmeI RE site:
GTT T AA AC
CAA ATT TG
Val STOP Thr
C-terminal tagged protein configuration: (SgfI GOI PmeI-Tag)
Protein expression starts from AUG start codon of GOI
Ends with stop codon of PmeI.
Take advantage of blunt end of PmeI and ligate with EcoICRI that converts
site to GTT-TCT-C (Val-Phe-X).
Side effect: ligation is permanent cant use PmeI as site has been
destroyed.
Homologous Recombination

Endonuclease creates nicks in one strand of DNA and crossing of two

strands take place thereby interchanging and giving recombinant DNA

Site specific recombination involves breakage and reunion of DNA

Here it recognises same sequence and cleaves them and two different
genes are ligated
Advantages of recombination cloning
Mediated by recombinases at site-specific sequences.Eliminates use of
REs and Ligases Extremely specific as sites are 30-200bp long so
extremely rare in genome Virtually any DNA fragment can be inserted
without worrying about interrupting the coding sequence. High efficiency
low reaction time. Do not require individual inspection of each target
sequence. Step 1: generation of master clone. Step 2: transfer to expression
clone.