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Reporter:Abby Guil G. OmelisMargie Lou Pajares

PCR PRIMER DESIGN

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!at is PCR Primer Desig"#It is $!ere small amou"ts o% t!ege"eti& material &a" "o$ beampli'e( to be able to a i(e"ti%y)

ma"ipulate DNA) (ete&t i"%e&tiousorga"isms) i"&lu(i"g t!e *iruses t!at&ause AIDS) !epatitis) tuber&ulosis)(ete&t ge"eti& *ariatio"s) i"&lu(i"gmutatio"s) i" !uma" ge"es a"("umerous ot!er tas+s.

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Steps %or Primer Desig",. De"aturatio"

- t!e ge"eti& material is (e"ature() &o"*erti"g t!e(ouble stra"(e( DNA mole&ules to si"gle stra"(s.. A""eali"g

- /!e primers are t!e" a""eale( to t!e&ompleme"tary regio"s o% t!e si"gle stra"(e(mole&ules.0. E1te"sio"

- t!ey are e1te"(e( by t!e a&tio" o% t!e DNApolymerase. All t!ese steps are temperaturese"siti*e a"( t!e &ommo" &!oi&e o% temperatures is23 oC) 45 oC a"( 65 oC respe&ti*ely.

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Steps %or PCR

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/!i"gs to &o"si(er %or PrimerDesig":

1. Primer Length: It is ge"erally a&&epte( t!att!e optimal le"gt! o% PCR primers is ,8- bp.2. Primer Melting Temperature: PrimerMelti"g /emperature 9/ m by (e'"itio" is t!e

temperature at $!i&! o"e !al% o% t!e DNA(uple1 $ill (isso&iate to be&ome si"glestra"(e( a"( i"(i&ates t!e (uple1 stability.3. Primer Annealing Temperature: /!e

primer melti"g temperature is t!e estimate o%t!e DNA-DNA !ybri( stability a"( &riti&al i"(etermi"i"g t!e a""eali"g temperature.

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/!i"gs to &o"si(er %or PrimerDesig":

4. GC Content5. GC Clamp6. Primer Secon ar!

Structure": Prese"&e o% t!e primerse&o"(ary stru&tures pro(u&e( byi"termole&ular or i"tramole&ulari"tera&tio"s &a" lea( to poor or "o yiel( o%

t!e pro(u&t.#. $epeat":%. $un"

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Parameters %or PrimerDesig":

1. Amplicon Length: /!e ampli&o" le"gt! is (i&tate(by t!e e1perime"tal goals.2. Pro uct Po"ition: Primer &a" be lo&ate( "ear t!e;< e"() t!e 0< e"( or a"y $!ere $it!i" spe&i'e(le"gt!.3. Tm o& Pro uct: Melti"g /emperature 9/ m is t!etemperature at $!i&! o"e !al% o% t!e DNA (uple1 $ill(isso&iate a"( be&ome si"gle stra"(e(.4. 'ptimum Annealing Temperature (T a 'pt*:

5. Primer Pair Tm Mi"match Calculation: /!e t$oprimers o% a primer pair s!oul( !a*e &losely mat&!e(melti"g temperatures %or ma1imi=i"g PCR pro(u&tyiel(.