Un genio bizzarro
-Kary Banks Mullis nasce il 28 dicembre 1944 a Lenoir,
North Carolina.
-Nel 1962 si diploma alla Dreher High School di
Columbia, South Carolina.
-Nel 1966 consegue il master in chimica al Georgia
institute of Thecnology.
-Nel 1972 consegue il Ph.D. in biochimica all’Università
della California a Berkeley.
-Nel 1973 ottiene una borsa di studio post-dottorato in
cardiologia pediatrica all’University of Kansas Medical
School.
-Nel 1977 si trasferisce all’Università della California per occuparsi di
chimica farmaceutica.
- Nel 1979 è assunto dalla Cetus Corporation di Emeryville, California, come
chimico esperto del DNA.
- Nel 1983 inventa la Reazione a Catena della Polimerasi per la quale nel
1993 gli sarà conferito il premio Nobel per la chimica.
- Nel 1986 viene nominato direttore per la Biologia Molecolare della
Xytronyx Inc. di San Diego (California).
-Attualmente è vicepresidente
per la Biologia Molecolare
presso la Burstein Techologies
con sede ad Irvine in
California.
Referenze
Saiki et al. (1985) Science 230:1350.
Saiki et al. (1986) Nature 324:163.
Mullis & Faloona (1987) Meth.
Enzymol. 155:335.
Erlish et al. (1988) Nature 331:461.
Letture consigliate
LE SCIENZE dossier
N. 15 - 2003
The polymerase chain
reaction (PCR)
The standard PCR reaction, typically
done in a volume of 50-100 ml,
contains:
- DNA template (102-105 copies)
- Appropriate buffer (50 mM KCl, 10
mM Tris-HCl pH 8)
- Magnesium (1,5-3 mM)
- dNTPs (50-200 mM each)
- Primers 20-30 bases in length (10-20
pmoles each)
- Taq DNA polymerase (1-2 Units)
DNA thermal cyclers
T gradient
Template DNA
molecules with a
constant-length end and
a variable-length end
Theoretical PCR amplification of a target fragment
with increasing number of cycles
N° = i x 2n
where:
i = initial number of DNA
molecules (duplex DNA with
constant 5’- and 3’-ends.)
n = number of cycles
Primer selection
-Select primers with a random base distribution and when possible with
reasonably high GC content.
-Avoid sequence with significant secondary structure.
-Check the primers against each other for complementarity.
MgCl2 (mM)
Effect of Mg2+ concentration
Generally excess of Mg2+ will
result in the accumulation of nonspecific amplification products
and insufficient Mg2+ will reduce
the yield.
1,8 Kb 
Units per 100 ml reaction
Effect of Taq polymerase
concentration
Generally the optimum
concentration of Taq polymerase
is 1-4 units per 100 ml reaction.
Increasing amounts can result in
greater production of non-specific
amplification products and
reduced yield of the desired target
fragment.
110 bp 
Cycling Parameters
Denaturation: DNA is denatured by briefly heating the
samples to 92-95 °C.
Annealing: the primers are allowed to anneal to their
complementary sequences by cooling to 40-60°C.
Because of the very large molar excess of primers present
in the reaction mix, hybridization occurs almost
instantaneously and long incubation at the annealing
temperature is not required (10-60 sec).
The melting temperature (Tm) of a PCR primer
C-G = 4°C
A-T = 2°C
A) 5’-GCGTTAGGCCAGCGGG-3’
B) 5’-CCATTTGAAATTTATA-3’
C) 5’-AGGTCCCCATCAGCGC-3’
Tm = 56°C
Tm = 38°C
Tm = 54°C
Extension: the annealed primers are extended with Taq
polymerase by heating to 70-75 °C. The time of incubation varies
according to the length of the target being amplified: allowing 1
min. for each kilobases of sequence is almost certainly excessive
(Taq polymerase can add up to 100-200 nucleotides per sec).
Nested PCR
If the primers bind to more than one locus (e.g. paralog or common
domain), then more than one segment of DNA will be amplified. To
control for these possibilities, investigators often employ nested
primers to ensure specificity.
Nested PCR means that two pairs of PCR primers were used for a
single locus. The first pair amplified the locus as seen in any PCR
experiment. The second pair of primers (nested primers) bind
within the first PCR product and produce a second PCR product
that will be shorter than the first one
First set of primers
and



Second set of primers
 and 



The logic behind this strategy is that if the wrong locus were amplified by
mistake, the probability is very low that it would also be amplified a second
time by a second pair of primers.
This procedure is designed to increase the sensitivity of PCR by directly
re-amplifying the product from a primary PCR with a second PCR.
Principali applicazioni della PCR
1)
Clonaggio di geni e screening di librerie genomiche
2)
Manipolazioni e sequenziamento del DNA
3)
DNA fingerprinting in medicina legale (test di paternità e di
identificazione di reperti biologici)
4)
Ricerca di OGM e tracciabilità degli alimenti
5)
Test per rilevare infezioni batteriche e virali (HIV,
Mycobacterium tubercolosis)
6)
Test per l’identificazione di oncogeni (ras)
7)
Studi di evoluzione molecolare (DNA antico)
8)
Studi del polimorfismo del DNA
Agarose gel electrophoresis of DNA
The gel slabs for DNA analysis are either agarose ( 0,6-2 %) or
polyacrylamide (5-12 %)
l DNA/HindIII
Analysis of plasmid DNA
L
S
Lanes 1,3 and 5 = undigested
DNA (S)
Lanes 2,4 and 6 = linearized
DNA (L)
When an electric current is passed through the gel, each fragment
moves down it at a rate that is inversely related to the log of its
molecular weight.
A DNA fragment
migrated 49 mm
Y = -0,026 X + 3,945
Y = -1,274 + 3,945
Y = 2,671
antilogY
469 bp
Agarobiose basic repeating
unit of AGAROSE
Agarose is derived from various species
of seaweed (Gracilaria or Gelidium).
The agarobiose repeat unit forms long
chains with an average molecular mass
of 120.000 Daltons. There are also
charged groups present on the
polysaccharide, most notably pyruvates
and sulphates
- The gelation process involves the formation of a
macroporous matrix (the pore size  100 - 300 nm
varies with concentration and type of agarose).
- Agarose gels are thermoreversible
(Melting T = 93°C ; Gelling T  30 - 40 °C)
DNA can be readily detected at high
sensitivity in a gel.
The bands of DNA in the gel are stained
with the intercalating dye ethidium
bromide, and as little as 10-20 ng of
DNA in one band can be detected as
visible fluorescence when the gel is
illuminated with UV light (  300 nm).
EtBr
EtBr