BIOCHEMISTRY

(Written Report) Jhonalyn J. Narvasa Prof.Biteng

Polymerase Chain Rea tion!PCR"

(Protocols)

Intro#$ tion

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene a part of a gene or a non!coding se"uence. #ost PCR methods typically amplify DNA fragments of up to $% &ilo base pairs (&b) although some techni"ues allo' for amplification of fragments up to (% &b in si)e. A basic PCR set up re"uires se*eral components and reagents. These components include+ DNA template that contains the DNA region (target) to be amplified. T'o primers 'hich are complementary to the DNA regions at the ,- (fi*e prime) or .- (three prime) ends of the DNA region. Ta" polymerase or another DNA polymerase 'ith a temperature optimum at around /%0C. Deo1ynucleoside triphosphates (dNTPs2 also *ery commonly and erroneously called deo1ynucleotide triphosphates) the building bloc&s from 'hich the DNA polymerases synthesi)es a ne' DNA strand. 3uffer solution pro*iding a suitable chemical en*ironment for optimum acti*ity and stability of the DNA polymerase. Di*alent cations magnesium or manganese ions2 generally #g45 is used but #n45 can be utili)ed for PCR!mediated DNA mutagenesis as higher #n45 concentration increases the error rate during DNA synthesis6/7 #ono*alent cation potassium ions.

The PCR is commonly carried out in a reaction *olume of $%!4%% 8l in small reaction tubes (%.4!%., ml *olumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achie*e the temperatures re"uired at each step of the reaction (see belo'). #any modern thermal cyclers ma&e use of the Peltier effect 'hich permits both heating and cooling of the bloc& holding the PCR tubes simply by re*ersing the electric current. Thin!'alled reaction tubes permit fa*orable thermal conducti*ity to allo' for rapid thermal e"uilibration. #ost thermal circlers ha*e heated lids to pre*ent condensation at the top of the reaction tube. 9lder thermocyclers lac&ing a heated lid re"uire a layer of oil on top of the reaction mi1ture or a ball of 'a1 inside the tube. There are many basic considerations to being an effecti*e PCR-er. :irst your laboratory needs to be properly e"uipped. The thermocycler may become the 'or&horse of the lab and can be used for PCR as 'ell as other en)ymatic reactions such as restriction en)ymes ligases polymerases. #a&e sure that any PCR machine you buy is fully programmable (can do the cycle profile as 'ell as certain ad*anced options such as ;touch!up; or ;touch!do'n; and increasing elongation time<cycle) can handle =>!'ell plates and has a heated lid for oil!free operation. ?n addition to ha*ing a PCR machine you 'ill also need to configure your lab space differently. @ou 'ill need a room dedicated to the assembly of PCR reactions and a separate lab for the e1amination of amplification products. NABAR ACC9W TD3AE C9NTA?N?NF A#PC?:?AD PCR PR9DDCTE T9 3A 9PANAD ?N TGA EA#A R99# AE TGAT DEAD T9 AEEA#3CA PCR RAACT?9NE. The best analogy regarding the problem of PCR contamination is to compare your lab space to that occupied by a s'imming pool. The electrophoresis chambers and PCR purification products are placed in a separate room from the thermocycler. Also you 'ill need dedicated pipetters for PCR assembly for PCR product handling and possibly for the handling of internal standards. The pipetters should be disassembled periodically and either u* irradiated or bleached. All tubes and pipette tips should be autocla*ed 'hile the tube retainer and trays should be bleached ($%H) after e*ery use. 9ther pieces of e"uipment necessary are a heat plate 'ith a =>!'ell bloc& set at I, C aerosol barrier pipet tips and a *orte1er con*eniently located ne1t to the thermocycler and of course electrophoresis units. 9ptional e"uipment include free)er bo1es for en)ymes a picofuge and if you are really paranoid about contamination use a PCR chamber 'ith a u* light. The second consideration is to familiari)e yourself 'ith the concepts of ho' the polymerase chain reaction 'or&s in general and specifically ho' "uantitati*e aspects are confused by tube!to!tube

*ariability. A Fuide to #ethods and Application of ho' PCR 'or&s is PCR protocols. The last item re"uired to be a good PCR-er is patience. This techni"ue is *ery sensiti*e to timing season *arious forms of contamination and intermittent bouts of stress and tedium. After a series of failures it is often best to 'al& a'ay from PCR and indulge in your fa*orite *ices for a period of time. Pro e#$re :igure $+ Echematic dra'ing of the PCR cycle

!%" &enat$ring at =(!=>0C.

!'" (nnealing at J>,0C

!)" Elongation at /40C.

:our cycles are sho'n here. The blue lines represent the DNA template to 'hich primers (red arro's) anneal that are e1tended by the DNA polymerase (light

green circles) to gi*e shorter DNA products (green lines) 'hich themsel*es are used as templates as PCR progresses. The PCR usually consists of a series of 4% to (% repeated temperature changes called cycles2 each cycle typically consists of 4!. discrete temperature steps. #ost commonly PCR is carried out 'ith cycles that ha*e three temperature steps (:ig. 4). The cycling is often preceded by a single temperature step (called hold) at a high temperature (K=%0C) and follo'ed by one hold at the end for final product e1tension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a *ariety of parameters. These include the en)yme used for DNA synthesis the concentration of di*alent ions and dNTPs in the reaction and the melting temperature of the primers. Initiali*ation ste++ This step consists of heating the reaction to a temperature of =(!=>0C (or =I0C if e1tremely thermostable polymerases are used) 'hich is held for $!= minutes. ?t is only re"uired for DNA polymerases that re"uire heat acti*ation by hot!start PCR.

&enat$ration ste++ This step is the first regular cycling e*ent and consists of heating the reaction to =(!=I0C for 4%!.% seconds. ?t causes melting of DNA template and primers by disrupting the hydrogen bonds bet'een complementary bases of the DNA strands yielding single strands of DNA.

(nnealing ste++ The reaction temperature is lo'ered to ,%!>,0C for 4%!(% seconds allo'ing annealing of the primers to the single!stranded DNA template. Typically the annealing temperature is about .!, degrees Celsius belo' the Tm of the primers used. Etable DNA!DNA hydrogen bonds are only formed 'hen the primer se"uence *ery closely matches the template se"uence. The polymerase binds to the primer!template hybrid and begins DNA synthesis.

E,tension-elongation ste++ The temperature at this step depends on the DNA polymerase used2 Ta" polymerase has its optimum acti*ity temperature at /,!I%0C 6$%76$$7 and commonly a temperature of /40C is used 'ith this en)yme. At this step the DNA polymerase synthesi)es a ne' DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in ,- to .- direction condensing the ,-!phosphate group of the dNTPs 'ith the .-!hydro1yl group at the end of the nascent (e1tending) DNA strand. The e1tension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule of!thumb at its optimum temperature the DNA polymerase 'ill

polymeri)e a thousand bases per minute. Dnder optimum conditions i.e. if there are no limitations due to limiting substrates or reagents at each e1tension step the amount of DNA target is doubled leading to e1ponential (geometric) amplification of the specific DNA fragment.

.inal elongation+ This single step is occasionally performed at a temperature of /%!/(0C for ,!$, minutes after the last PCR cycle to ensure that any remaining single!stranded DNA is fully e1tended.

.inal hol#+ This step at (!$,0C for an indefinite time may be employed for short!term storage of the reaction. PCR stages The PCR process can be di*ided into three stages+ A1ponential amplification+ At e*ery cycle the amount of product is doubled (assuming $%%H reaction efficiency). The reaction is *ery specific and precise. Ce*elling off stage+ The reaction slo's as the DNA polymerase loses acti*ity and as consumption of reagents such as dNTPs and primers causes them to become limiting. Plateau+ No more product accumulates due to e1haustion of reagents and en)yme.

PCR o+timi*ation PCR can fail for *arious reasons in part due to its sensiti*ity to contamination causing amplification of spurious DNA products. 3ecause of this a number of techni"ues and procedures ha*e been de*eloped for optimi)ing PCR conditions. Contamination 'ith e1traneous DNA is addressed 'ith lab protocols and procedures that separate pre!PCR mi1tures from potential DNA contaminants. This usually in*ol*es spatial separation of PCR!setup areas from areas for analysis or purification of PCR products and thoroughly cleaning the 'or& surface bet'een reaction setups. Primer!design techni"ues are important in impro*ing PCR product yield and in a*oiding the formation of spurious products and the usage of alternate buffer components or polymerase en)ymes can help 'ith amplification of long or other'ise problematic regions of DNA.

.a tors (ffe ting the PCR Denaturing Temperature and time The specific complementary association due to hydrogen bonding of single!stranded nucleic acids is referred to as ;annealing;+ t'o complementary se"uences 'ill form hydrogen bonds bet'een their complementary bases (F to C and A to T or D) and form a stable double!stranded anti!parallel ;hybrid; molecule. 9ne may ma&e nucleic acid (NA) single!stranded for the purpose of annealing ! if it is not single!stranded already li&e most RNA *iruses ! by heating it to a point abo*e the ;melting temperature; of the double! or partially!double!stranded form and then flash!cooling it+ this ensures the ;denatured; or separated strands do not re! anneal. Additionally if the NA is heated in buffers of ionic strength lo'er than $,%m# NaCl the melting temperature is generally less than $%%oC ! 'hich is 'hy PCR 'or&s 'ith denaturing temperatures of =$!=/oC. Ta" polymerase is gi*en as ha*ing a half!life of .% min at =,oC 'hich is partly 'hy one should not do more than about .% amplification cycles+ ho'e*er it is possible to reduce the denaturation temperature after about $% rounds of amplification as the mean length of target DNA is decreased+ for templates of .%%bp or less denaturation temperature may be reduced to as lo' as IIoC for ,%H (F5C) templates (@ap and #cFee $==$) 'hich means one may do as many as (% cycles 'ithout much decrease in en)yme efficiency. ;Time at temperature; is the main reason for denaturation < loss of acti*ity of Ta"+ thus if one reduces this one 'ill increase the number of cycles that are possible 'hether the temperature is reduced or not. Normally the denaturation time is $ min at =(oC+ it is possible for short template se"uences to reduce this to .% sec or less. ?ncrease in denaturation temperature and decrease in time may also 'or&+ ?nnis and Felfand ($==%) recommend =>oC for $, sec.

(nnealing Tem+erat$re an# Primer &esign Primer length and se"uence are of critical importance in designing the parameters of a successful amplification+ the melting temperature of a NA duple1 increases both 'ith its length and 'ith increasing (F5C) content+ a simple formula for calculation of the Tm is Tm L ((F 5 C) 5 4(A 5 T)oC. Thus the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). 9ne should aim at using an annealing temperature (Ta) about ,oC belo' the lo'est Tm of ther pair of primers to be used

(?nnis and Felfand $==%). A more rigorous treatment of Ta is gi*en by Rychli& et al. ($==%)+ they maintain that if the Ta is increased by $oC e*ery other cycle specificity of amplification and yield of products M$&b in length are both increased. 9ne conse"uence of ha*ing too lo' a Ta is that one or both primers 'ill anneal to se"uences other than the true target as internal single!base mismatches or partial annealing may be tolerated+ this is fine if one 'ishes to amplify similar or related targets2 ho'e*er it can lead to ;non!specific; amplification and conse"uent reduction in yield of the desired product if the .-!most base is paired 'ith a target. A conse"uence of too high a Ta is that too little product 'ill be made as the li&elihood of primer annealing is reduced2 another and important consideration is that a pair of primers 'ith *ery different Tas may ne*er gi*e appreciable yields of a uni"ue product and may also result in inad*ertent ;asymmetric; or single!strand amplification of the most efficiently primed product strand. Annealing does not ta&e long+ most primers 'ill anneal efficiently in .% sec or less unless the Ta is too close to the Tm or unless they are unusually long.

Primer /ength The optimum length of a primer depends upon its (A5T) content and the Tm of its partner if one runs the ris& of ha*ing problems such as described abo*e. Apart from the Tm a prime consideration is that the primers should be comple1 enough so that the li&elihood of annealing to se"uences other than the chosen target is *ery lo'. (Eee hybridn.doc). :or e1ample there is a N chance ((!$) of finding an A F C or T in any gi*en DNA se"uence2 there is a $<$> chance ((!4) of finding any dinucleotide se"uence (eg. AF)2 a $<4,> chance of finding a gi*en (!base se"uence. Thus a si1teen base se"uence 'ill statistically be present only once in e*ery ( $> bases (L( 4=( =>/ 4=> or ( billion)+ this is about the si)e of the human or mai)e genome and $%%%1 greater than the genome si)e of A. coli. Thus the association of a greater! than!$/!base oligonucleotide 'ith its target se"uence is an e1tremely se"uence! specific process far more so than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Conse"uently $/!mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer length may mean that e*en high annealing temperatures are not enough to pre*ent mismatch pairing and non!specific priming.

&egenerate Primers :or amplification of cognate se"uences from different organisms or for ;e*olutionary PCR; one may increase the chances of getting product by designing

;degenerate; primers+ these 'ould in fact be a set of primers 'hich ha*e a number of options at se*eral positions in the se"uence so as to allo' annealing to and amplification of a *ariety of related se"uences. :or e1ample Compton ($==%) describes using $(!mer primer sets 'ith ( and , degeneracies as for'ard and re*erse primers respecti*ely for the amplification of glycoprotein 3 (g3) from related herpes*iruses. The re*erse primer se"uence 'as as follo's+ TCFAATTCNCC@AA@TFNCCNT 'here @ L T 5 C and N L A 5 F 5 C 5 T and the I!base ,-!terminal e1tension comprises a AcoR? site (underlined) and flan&ing spacer to ensure the restriction en)yme can cut the product (the Ne' Angland 3iolabs catalogue gi*es a good list of 'hich en)ymes re"uire ho' long a flan&ing se"uence in order to cut stub ends). Degeneracies ob*iously reduce the specificity of the primer(s) meaning mismatch opportunities are greater and bac&ground noise increases2 also increased degeneracy means concentration of the indi*idual primers decreases2 thus greater than ,$4!fold degeneracy should be a*oided. Go'e*er ? ha*e used primers 'ith as high as 4,>! and $%4(!fold degeneracy for the successful amplification and subse"uent direct se"uencing of a 'ide range of Mastrevir$ses against a bac&ground of mai)e genomic DNA.

Primer se"uences 'ere deri*ed from multiple se"uence alignments2 the mismatch positions 'ere used as (!base degeneracies for the primers (sho'n as stars2 , in : and ( in R) as sho'n abo*e. Despite their degeneracy the primers could be used to amplify a 4,% bp se"uence from *iruses differing in se"uence by as much as ,%H o*er the target se"uence and >%H o*erall. They could also be used to *ery sensiti*ely detect the presence of #ai)e strea& *irus DNA against a bac&ground of mai)e genomic DNA at dilutions as lo' as $<$%= infected sap < healthy sap (see belo').

Eome groups use deo1yinosine (d?) at degenerate positions rather than use mi1ed oligos+ this base!pairs 'ith any other base effecti*ely gi*ing a four!fold degeneracy at any postion in the oligo 'here it is present. This lessens problems to do 'ith depletion of specific single oligos in a highly degenerate mi1ture but may result in too high a degeneracy 'here there are ( or more d?s in an oligo. Alongation Temperature and Time This is normally /% ! /4oC for %., ! . min. Ta" actually has a specific acti*ity at ./oC 'hich is *ery close to that of the Oleno' fragment of A coli DNA polymerase ? 'hich accounts for the apparent parado1 'hich results 'hen one tries to understand ho' primers 'hich anneal at an optimum temperature can then be elongated at a considerably higher temperature ! the ans'er is that elongation occurs from the moment of annealing e*en if this is transient 'hich results in considerably greater stability. At around /%oC the acti*ity is optimal and primer e1tension occurs at up to $%% bases<sec. About $ min is sufficient for reliable amplification of 4&b se"uences (?nnis and Felfand $==%). Conger products re"uire longer times+ . min is a good bet for .&b and longer products. Conger times may also be helpful in later cycles 'hen product concentration e1ceeds en)yme concentration (K$n#) and 'hen dNTP and < or primer depletion may become limiting. Cy le N$m0er The number of amplification cycles necessary to produce a band *isible on a gel depends largely on the starting concentration of the target DNA+ ?nnis and Felfand ($==%) recommend from (% ! (, cycles to amplify ,% target molecules and 4, ! .% to amplify .1$%, molecules to the same concentration. This non! proportionality is due to a so!called plateau effect 'hich is the attenuation in the e1ponential rate of product accumulation in late stages of a PCR 'hen product reaches %.. ! $.% n#. This may be caused by degradation of reactants (dNTPs en)yme)2 reactant depletion (primers dNTPs ! former a problem 'ith short products latter for long products)2 end!product inhibition (pyrophosphate formation)2 competition for reactants by non!specific products2 competition for primer binding by re!annealing of concentrated ($%n#).

cDNA PCR A *ery useful primer for cDNA synthesis and cDNA PCR comes from a se"uencing strategy described by Th'eatt et al. ($==%)+ this utilised a mi1ture of three 4$!mer primers consisting of 4% T residues 'ith .-!terminal A F or C respecti*ely to se"uence inside the poly(A) region of cDNA clones of mRNA from eu&aryotic origin. ? ha*e used it to amplify discrete bands from a *ariety of poly(A)5 *irus RNAs 'ith only a single specific degenerate primer upstream+ the T!primer may anneal any'here in the poly(A) region but only molecules 'hich anneal at the beginning of the poly(A) tail and 'hose .-!most base is complementary to the base ne1t to the beginning of the tail 'ill be e1tended. eg+ ,-!TTTTTTTTTTTTTTTTTTTTTTTTT(A F C)!.A simple set of rules for primer sequence design is as follows :primers should be 17-28 bases in length; $. base composition should be ,%!>%H (F5C)2 4. primers should end (.-) in a F or C or CF or FC+ this pre*ents ;breathing; of ends and increases efficiency of priming2 3. Tms bet'een ,,!I%oC are preferred2 (. runs of three or more Cs or Fs at the .-!ends of primers may promote mispriming at F or C!rich se"uences (because of stability of annealing) and should be a*oided2 5. .-!ends of primers should not be complementary (ie. base pair) as other'ise primer dimers 'ill be synthesised preferentially to any other product2 6. primer self!complementarity (ability to form 4o structures such as hairpins) should be a*oided. /a0elling PCR Pro#$ ts 1ith &igo,igenin

PCR products may be *ery con*eniently labelled 'ith digo1igenin!$$! dDTP (3oehringer!#annheim) by incorporating the reagent to $%!.,H final effecti*e dTTP concentration in a nucleotide mi1 of final concentration ,%!$%%u# dNTPs (Amanual $==$2 Nucleic Acids Res $=+ 4/=%). This allo's substitution to a &no'n e1tent of probes of e1actly defined length 'hich in turn allo's e1actly defined bybridisation conditions. ?t is also the most effecti*e means of labelling PCR products as it is potentially unsafe and BAR@ e1pensi*e to attempt to do similarly 'ith .4P!dNTPs and nic&!translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling. Cleaning PCR Pro#$ ts

Fetting rid of mineral oil+ simply add ,%ul of chloroform to the reaction *ial *orte1 and centrifuge briefly and remo*e the ;hanging droplet; of APDA9DE solution 'ith a micopipette. Fetting rid of 'a1 or Baseline+ simply ;spear; 'a1 gem and remo*e2 do as for oil or bottom!puncture tube and blo' out a"ueous drop for Baseline. Cleaning!up DNA+ . options a protocol 'hich gi*es DNA that is clean enough for se"uencing is the follo'ing+ increase *olume to $%%ul 'ith 'ater add $%# ammonium acetate soln. to %.4# final concentration ($<,th *olume) add e"ual *olume of isopropanol (propan!4! ol) lea*e on bench , min centrifuge 4% min at $, %%% rpm remo*e li"uid using pipette resuspend in $%%ul 'ater or TA repeat precipitation. Eimply do a phenol!CGCl. e1traction (add 4%ul phenol to a"ueous phase *orte1 add ,%ul CGCl. *orte1 centrifuge remo*e DPPAR a"ueous phase repeat CGCl. e1traction). #a&e a"ueous phase up to (%%ul and spin through #illipore Dltrafree!#C N#WC .% %%% cartridges (at >%%% rpm in microcentrifuge) 'ash through 'ith 41(%%ul 'ater collect 5<!4%ul sample+ this is pure enough for se"uencing. Se2$en ing PCR Pro#$ ts

This is best done using ssDNA generated by asymmetric PCR and the ;limiting; primer for se"uencing. Go'e*er efficient se"uencing of dsDNA generated by normal PCR is possible using the modification to the Ee"uenaseT# protocol published by 3achmann et al. ($==%) (NAR $I+ $.%=). CCAAN DNA is resuspended in se"uencing buffer containing %.,H Nonidet P!(% and %.,H T'een!4% and se"uencing primer denatured by heating to =,oC for , min snap!cooled on 'et ice and se"uenced by the ;close!to!primer; protocol (eg+ dilute e1tension mi1es).

Cloning PCR Pro#$ ts

T!A Cloning Etrategy+ Ta" and other polymerases seem to ha*e a terminal transferase acti*ity 'hich results in the non!templated addition of a single nucleotide to the .-!ends of PCR products. ?n the presence of all ( dNTPs dA is preferentially added2 ho'e*er use of a single dNTP in a reaction mi1 results in (relati*ely inefficient) addition of that nucleotide. This complicates cloning as the supposedly blunt!ended PCR product often is not and blunt!ended cloning protocols often do not 'or& or are *ery inefficient. This can be remedied by incubation of PCR products 'ith T( DNA pol or Oleno' pol 'hich ;polishes; the ends due to a .-!K,e1onuclease acti*ity (Cui and Ech'art) $==42 3ioTechni"ues 4%+ 4I!.%). Go'e*er this terminal transferase acti*ity is also the basis of a cle*er cloning strategy+ this uses Ta" pol to add a single dT to the .-!ends of a blunt!cut cloning *ector such as pDC or p3luescriptT# and simple ligation of the PCR product into the no' ;stic&y! ended; plasmid (#archu& et al. $==%2 NAR $=+ $$,>). In or+oration of Restri tion Sites in Primers

Although this may be rendered simple by incorporating the same or different restriction sites at the ,-!ends of PCR primers ! 'hich allo's generation of stic&y ends and straightfor'ard cloning into appropriate *ectors ! these should ha*e AT CAAET t'o additional bases ,- to the recognition se"uence to ensure that the en)ymes 'ill in fact recognise the se"uence ! and it is often found that e*en 'hen this is done the efficiency of cutting of fresh product is ne1t to )ero. This can sometimes be remedied by incubating fresh product 'ith Proteinase O (to digest off tightly!attached Ta" pol) but often is not. A solution to the problem is to use the ;Oleno'!Oinase!Cigase; (OOC) method+ this in*ol*es ;polishing; products 'ith Oleno' &inasing them to get ,-!phosphorylation (N3+ 9C?F9NDCCA9T?DA PR?#ARE N9R#ACC@ GABA N9 ,-!PG9EPGATAEQQQ) ligating the fragments together to get concatemers then restricting these 'ith the appropriate restriction en)ymes to generate the stic&y!ended fragments suitable for cloning (Corens $==$2 PCR #ethods and Applications $+ $(%!$($). (N& (/3(YS REMEMBER

W9RO CCAAN T?TRATA #AFNAE?D# D9N-T DEA T99 #DCG TA#PCATA DNA D9N-T DEA PCR PR9DDCTE ?N PCR PRAPARAT?9N ARAAE ACWA@E ACWA@E ?NCCDDA WATAR AND BAR@ D?CDTA P9E?T?BA ABAR@ ARPAR?#ANT WAAR FC9BAE DEA PCDFFAD T?PE

C9NTR9CE

?N