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`SKIExhibit
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`2068Page1of42
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`Page 1 of 42
`SKI Exhibit 2068
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`Page 2 of 42T
`SKI Exhibit 2068
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`The INTRODUCTION TO BIOTECHNIQUESseries
`
`Editor:
`
`CENTRIFUGATION
`RADIOISOTOPES
`LIGHT MICROSCOPY
`ANIMAL CELL CULTURE
`GEL ELECTROPHORESIS: PROTEINS
`MICROBIAL CULTURE
`ANTIBODY TECHNOLOGY
`GENE TECHNOLOGY
`LIPID ANALYSIS
`GEL ELECTROPHORESIS: NUCLEIC ACIDS
`LIGHT SPECTROSCOPY
`PCR, SECOND EDITION
`
`Page 3 of 42
`
`D. Billington School of Biomolecular Sciences, Liverpool John
`Moores University, Byrom Street, Liverpool L3 3AF
`
`Forthcomingtitles
`
`PLANT CELL CULTURE
`MEMBRANE ANALYSIS
`
`SKI Exhibit 2068
`SKI Exhibit 2068
`Page 3 of 42
`
`

`

`PCR
`
`Second Edition
`
`C.R. Newton and A. Graham
`
`Page 4 of 42
`
`ZENECA Pharmaceuticals, Mereside, Alderley Park, Macclesfield,
`Cheshire SK10 4TG, UK
`
`Bios
`atpli
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`& |Springer
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`
`SKI Exhibit 2068
`SKI Exhibit 2068
`Page 4 of 42
`
`

`

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`$907HIGIUXAIMS
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`SKI Exhibit 2068
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`Page 10 of 42T
`SKI Exhibit 2068
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`SKI Exhibit 2068
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`Page 13 of 426U
`SKI Exhibit 2068
`
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`

`

`PCRwas invented in 1985 by Kary Mullis [1], then working for Cetus
`Corporation in California, while driving late at night pondering new
`waysto detect specific bases by DNA sequencing. Indeed, Kary Mullis
`gives us a fascinating insight into what led to this invention in a
`Scientific American paper[2]. The 1993 Nobel prize for chemistry was
`awarded to Dr Mullis for having invented PCR. Theoriginal protocols
`for PCR amplification [1,3,4] used the Klenow fragment of E. coli
`SONA
`DNApolymeraseI to catalyze the oligonucleotide extension. However,
`this enzymeis thermallyinactivated during the denaturation step of
`a PCR cycle andsothereséarchers had to add a fresh aliquot of
`enzymeat each cycle of the amplification process. Obviously this was
`a serious limitatienpespecially to attempts to automate the technique.
`
`Page 14 of 42
`
`Molecules
`
`Molarity
`
`Weight
`
`Molecules
`
`325Da)
`ofadNMPIs
`larweight
`
`
`
`1.33x10"
`
`8,01x10°
`
`
`
`=48500bp)
`
`60000unitsa
`
`meraseactivity
`
`PCR
`
`WHATIS PCR?
`
`omponents -
`
`DONAtemplate
`DNApolymerase
`dNTPs
`Primers
`Buffer
`
`Thermal cycler
`for amplification
`
`FIGURE 1.5: Basic components of a PCR amplification. Template DNA,
`buffer, dNTPs and primers are mixed and heated to denature the DNA
`thermally. DNA polymerase is added and mixed. The reaction components
`are overlaid with mineral oil and the tube placed in a thermalcycler. At the
`endof the amplification, the products are analyzed to confirm that the PCR
`has been successful.
`
`such as buffers, MgCl, and dNTPsare also available from many
`suppliers or can be prepared by the researcher. A wide range of
`thermostable DNA polymerases is now available from many
`manufacturers and the applications and properties of these are
`discussed in moredetail in Section 2.2.
`
`The use of the(Klenoensyme worked well for the amplification of
`
`DNA (<200 bp), but was disappointing for larger
`short fragments of
`amplicons. Often the yields were low and the products showedsize
`heterogeneity [5]. This was probably due to the low annealing and
`extension temperatures (37°C) which had to be used for the Klenow
`enzymeto be catalytically active. Another serious limitation in the
`early days of the PCR technique was the use of manualtransfer of
`samples between water baths or heating blocks maintained at the
`
`SKI Exhibit 2068
`SKI Exhibit 2068
`Page 14 of 42
`
`&$ 8=aEP
`
`s2 o™o & 3&Sa53%a= >& S=«w
`
`y-
`
`<- Before
`
`

`

`8
`
`PCR
`
`specific temperatures required for each PCR cycle. Two crucial
`technological advances have made PCR such a key procedure in
`research andclinical laboratories:
`
`(i)
`
`the use of thermostable DNA polymerases which greatly simplify
`the whole procedure because the addition of fresh enzyme after
`each denaturation step is no longer
`required. The first
`thermostable DNA polymerase to be introduced into PCRs was
`Taq DNA polymerase(see Section 2.2.1) [6];
`(ii) the development of a variety of simple temperature cycling
`devices (also known as thermal cyclers or PCR machines) which
`led to the automation of the PCR (see Section 2.1).
`
`2 Instrumentation,
`Reagents and
`Consumables
`
`The Polymerase Chain Reaction (PCR) process is covered by patents
`owned by Hoffmann-La RocheInc. and F. Hoffman-La Roche,Ltd.
`
`
`
`References
`
`. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and
`Arnheim, N. (1985) Science, 230, 1350.
`. Mullis, KB. (1990) Scientific American, April 1990, 56.
`. Mullis, K.B., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H.A. (1986) Cold
`Spring Harbor Symp. Quant. Biol., 51, 263.
`. Mullis, KB. and Faloona, F. (1987) Methods Enzymol., 155, 335.
`. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Science, 233, 1076.
`5. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis,
`KB. and Erlich, H.A. (1988) Science, 239, 487.
`
`2.1 Instruments
`
`In the evolution of the PCR two developments have greatly
`contributed to the success of the procedure: the use of a thermostable
`DNApolymerase (see Section 2.2) and instrumentation to allow the
`automation of temperature cycling. Two types of approaches to
`automating PCR have been devised, based on robotics or temperature
`cycling devices. In robotics methods the samples are mechanically
`moved between controlled temperature positions. There are
`disadvantages with this approachparticularly in the large amountof
`bench space such instruments occupy, slow temperature equilibration
`if fine tuning is necessary and the difficulty of controlling
`temperature change when samples are being transported. Therefore
`temperature cycling devices have been the choice for PCR.
`
`Page 15 of 42
`
`Several designs of temperature cycling instruments have been used,
`for example:
`(i) heating and cooling byfluids;
`(ii) heating by electric resistance and cooling by fluid (e.g. tap-water
`or
`recycling water
`in a refrigerated bath) or
`refrigerant
`circulation;
`(iii)heating by electric resistance and cooling by semiconductors
`(Peltier devices);
`(iv) heating matt black metal surfaces bylight followed byair cooling.
`Thermal cycling devices are nowavailable from many manufacturers
`(see Appendix B) and these have easy-to-use software for
`fully
`automated operation,
`together with accurate and reproducible
`
`9
`
`SKI Exhibit 2068
`SKI Exhibit 2068
`Page 15 of 42
`
`

`

`10
`
`PCR
`
`INSTRUMENTATION, REAGENTS, CONSUMABLES
`
`11
`
`
`
`temperaturecontrol in all sample positions in the cycler. A typical
`temperature cycling profile for a three-step protocol
`is shown in
`Figure 2.1. The thermalcycling parametersarecritical to a successful
`PCR.The following parametersare listed as a guide to the important
`steps in the temperature and timeprofile of thermal cycling:
`* denaturation,
`* annealing of primers,
`* extension of the primers,
`* cycle number,
`* ramp times.
`Initial denaturation of template DNA at 95-100°C is sufficient to
`completely denature complex genomic DNAso that the primers can
`anneal after cooling. Thermal damage of DNAleads to an increased
`nucleotide misincorporation rate during PCR and high temperatures
`should be avoidedif high fidelity is required [1]. However, supercoiled
`
`= Mineraloil
`7"PCA
`1007
`go- 4
`
`One cycle. (repeat for n cycles)
`
`;
`
`—_.
`
`Te
`4
`S 704
`a
`=
`60- =
`2
`a Step 3
`5
`50>
`E
`|
`Swap 2
`4075
`6
`
`|
`F
`30
`0
`2
`4
`6
`8 10
`12 14 16 18 20
`Time (min)
`
`FIGURE 2.1: PCR temperature cycling profile. Step 1: Heat denature the
`double-stranded DNA in the presence of primers, the four dNTPs, PCR
`buffer and a thermostable DNA polymerase. Denaturation is normally in
`the range 93-100°C. Step 2: Annealthe oligonucleotide primers to the
`denatured template by lowering the temperature to 37-65°C depending on
`the T,, of the oligonucleotide primers. Step 3: Extend the primers at 72°C
`with a thermostable DNA polymerase. Steps 1 to 3 constitute one cycle of
`PCR. This process is then normally repeated (cycled) for at least 20 cycles.
`At the last cycle the extension time may be increased by several minutes
`to ensure the complete synthesis ofall strands.
`
`plasmid DNA substrates require boiling for several minutes to
`achieve complete denaturation. Denaturation during the PCR (i.e.
`cycle 2 onwards) is usually sufficient at 92-95°C but should be
`determined empirically as different PCR templates, thermal cyclers
`and tubes may have different requirements.
`
`The primer annealing step is an important parameter in optimizing
`the specificity of a PCR,andcalculation of
`#~/annealing
`temperature
`is described in Section 2.4.2. The calculated annealing temperatureis
`used as a starting point
`for experimental work; however,
`the
`annealing temperature should also be optimized empirically.
`
`Primerextension is usually performed at 72°C, whichis the optimum
`temperature for Tag/Amplitaq® DNA polymerase. The duration of the
`extension steps can be increased if long amplicons are being
`amplified, but for the majority of PCRs times of up to 2 min are
`usually sufficient. Often the extension timeofthefinalcycle is longer
`(up to 10 min) in an attempt to ensure that all product molecules are
`fully extended.
`a LA,
`The numberofcycles is usually between 25 and 35. With increasing
`cycle numbers it is commonto observe an increase in the amountof
`unwantedartifactual products and noincrease in the desired product.
`Therefore it is unusual to find reactions that use more than 40 cycles.
`
`The ramp time is that taken to change from one temperatureto the
`next in the thermal cycler, and is dependent on the type of equipment
`used. Generally, the fastest ramps which can be attained are used for
`POR. The actual ramp times can be determined by measuring the
`sample temperature with a thermocouple probe.
`
`Tt is also possible to use a two-step protocol for PCR where the
`annealing and primer extension steps are both performed at a
`temperature up to 72°C and the denaturation step at 92-100°C. The
`temperature at which annealing and extension has to be performed is
`a compromise between the primers being able to anneal and the
`polymerase being sufficiently active to perform the extension.
`Recent innovations in thermal cyclers include:
`(i)
`the use of 96-well microtiter formats, in place of microcentrifuge
`tubes, which allows robotic liquid handling devices to be used in
`sample preparation; 384-well plate thermocyclers are also
`available from Biometra;
`(ii) the availability of temperature cyclers which allow different
`thermal cycles to be performed in independent blocks on the same
`machine;
`
`SKI Exhibit 2068
`SKI Exhibit 2068
`Page 16 of 42
`
`Page 16 of 42
`
`

`

`12
`
`PCR
`
`INSTRUMENTATION, REAGENTS, CONSUMABLES
`
`13
`
`TABLE 2.1: Thermostable DNA polymerases andtheir sources
`DNA polymerase
`Natural/Recombinant
`Source
`Taq
`Natural
`Thermus aquaticus
`Amplitag*
`Recombinant
`T. aquaticus
`Amplitag® (Stoffel
`Recombinant
`T. aquaticus
`fragment)
`Hot Tub™
`Natural
`Thermusflavis
`Pyrostase™
`Natural
`T. flavis
`Pfu
`Natural
`Pyrococcusfuriosus
`Pwo
`Natural
`Pyrococcus woesei
`Tbr
`Natural
`Thermus brockianus
`Tf
`Natural
`T. flavis
`Thi
`Recombinant
`Thermococcus|itoralis
`Vent™
`Recombinant
`T. litoralis
`DeepVent™
`Recombinant
`Pyrococcus GB-D
`Tth
`Recombinant
`Thermus thermophilus
`
`UlTma™ Thermotoga maritima Recombinant
`
`
`TABLE 2.2: Properties of the DNA polymerases commonly used in PCR
`
`
`
`Stoffel
`Taq!
`Amplitaq* fragment DeepVent™ Vent™ Pfu Tth UlTma™
`
`Yes
`
`No
`
`Thermostability; 40
`half-life at 95°C
`(min)
`Say
`exonuclease
`activity
`5’
`exonuclease
`activity
`Processivity
`Extension rate
`(nt sec’)
`Reverse
`transcriptase
`activity
`3’A
`Resulting DNA
`ends
`ni.
`Strand
`displacement
`Molecular weight 94
`(kDa)
`
`80
`
`No
`
`No
`
`5-10
`>50
`Weak
`
`vA
`ni.
`61
`
`400
`
`No
`
`Yes
`
`7
`>80
`ni.
`
`>95%
`blunt
`Yes’
`ni.
`
`1380
`
`>120 20
`
`>50°
`
`No
`
`Yes
`
`ni.
`ni.
`ni.
`
`No Yes No
`
`Yes No
`
`Yes
`
`30-40 nai.
`ni.
`>33
`ni.
`60
`ni, Yes
`ni,
`
`>95% ni 3A Blunt
`blunt
`Yes*
`oni. mi.
`mi.
`ni.
`92
`94
`70
`
`
`
`2.2 Enzyme choice
`
`The introduction of thermostable DNA polymerases for PCR was one
`of the major technological advances which made PCR such a routine
`technique in research and clinical
`laboratories. Before the
`introduct