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 µl,
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 µM 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 µl reaction
Effect of Taq polymerase
concentration
Generally the optimum
concentration of Taq polymerase
is 1-4 units per 100 µl 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’
Tm = 56°C
B) 5’-CCATTTGAAATTTATA-3’
Tm = 38°C
C) 5’-AGGTCCCCATCAGCGC-3’
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
4
4
3
Second set of primers
4 and 3
3
4
3
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.
Quantitative Comparative PCR
Quantitative comparative PCR (Q/C PCR) utilizes an exogenously
added internal standard, which is composed of natural DNA fragments,
flanked by a common target sequence. In a competitive PCR, one set
of primers (directed to the target gene) is used to amplify both the
target gene and the natural DNA fragment. The DNA fragments
compete with the target DNA for the same primers and thus act as an
internal standard. The internal standard is designed to generate a PCR
product of a different size than the target gene (e.g. a 60-80bp
longer/shorter DNA product resulting from a manipulation of the gene of
interest by DNA engineering). The Q/C PCR reaction targets two
templates competing for the same primers in the same reaction.
By knowing the amount of internal control added to the reaction, one
can determine the amount of target template in the reaction.
Copy number/µ
µg DNA
Internal control
Target DNA
Product from Q/C PCR amplification of ten fold serial dilutions
of internal control, natural DNA and the target gene.
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
Tandem Nucleotide Repeat Markers
Minisatellites, 10-100 bp repeated several times in tandem (Variable Number
Tandem Repeats)
Microsatellites, 2-4 bp repeated several times in tandem (Short Tandem Repeat
Polymorphisms)
Tandem nucleotide repeat are
generated by slippage mutation
occurring during DNA replication
PCR amplification
of microsatellite
markers
Random Amplified
Polymorphic DNA
(RAPD)
In human genome
(3x109) a random
10 nucleotide
primer should be
represented about
3000 times
DNA fingerprinting in Forensic Science
In most of the cases, DNA fingerprinting is based
on minisatellite or microsatellite markers.
The DNA markers in a
DNA fingerprinting
are inherited
Screening of clones
father
child
child
DNA fingerprinting
in Paternity Testing
mother
When different probes are used to make several
fingerprints, the likelihood that any two
individuals chosen at random will have identical
matches in all of them is extremely small (less
than 1 in 1 trillion).
Diagnosi prenatale e
determinazione del sesso
La sequenza DYZ1 (3,5 Kb) è
presente nel cromosoma Y in ben
5000 copie. Mediante PCR (60-80
cicli) si amplifica un frammento di
154 bp che è peculiare dei maschi
(individuazione di malattie
ereditarie legate al cromosoma X).
Y-specific primers (154 bp)
Alu control primers (130 bp)
Ladder
Samples
154 bp →
1
2
3 4
5 L
1
2 3
4
5
← 130 bp
PCR asimmetrica
Questa applicazione della PCR è usata comunemente in
laboratorio per il sequenziamento del DNA
G A TC
G A TC
Sequence was determined form singlestranded DNA generated by
asymmetric PCR from either a DNA
PCR (from cDNA) (A) or RT-PCR
(from RNA extracted) (B).
Sanger Dideoxy Sequencing (1977)
Identificazione OGM
1) Estrazione del DNA
2) Amplificazione DNA
- Si amplifica il promotore 35S (virus del mosaico del cavolfiore) e/o il
terminatore nos (presenti negli alimenti contenenti OGM)
- si amplificano anche altri geni di controllo (C) per valutare l’amplificabilità
del DNA estratto (es: lectina della soia, la zeina del mais, il tRNA della
leucina per i vegetali)
OGM+
OGMS os
s
S
35 No C 35 N C
3) Controllo degli
amplificati su gel di
agarosio
4) Analisi dei risultati
Se lo screening è positivo si effettua una
PCR specifica per identificare con
precisione di quale OGM si tratti ( es. mais
Bt-176 o soia Roundup Ready ecc.)
PCR Quantitativa
Multiplex PCR allows analysis of two or more targets simultaneously. This
PCR technique is used for genetic screening, microsatellite analysis, and other
applications where it is necessary to amplify several products in a single
reaction
In this assay multiplex PCR was conducted with primers specific for 8
exons and the promoter region of human dystrophin gene (N° of expected
fragments = 9) Note the PCR band missing at 410 bp, possibly indicating a
deletion
Agarose gel electrophoresis of DNA
The gel slabs for DNA analysis are either agarose ( 0,6-2 %) or
polyacrylamide (5-12 %)
λ 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