nested touch down PCR

33 2 () V o l. 33 N o. 2 2005 2 Jour. of N o rthw est Sci2T ech U niv. of A gri. and Fo r. (N aṫ Sci. Ed. ) Feb. 2005 nested touch down PCR CAPN1 EST Ξ 1, 2, 3, 2, 4, 3 (1, 712100; 2, 730070; 3, 100083; 4, 748300) [ ] 1 (CA PN 1) GeneBank CA PN 1 cdna PCR, R T, nested touch2dow n PCR RNA, pgem 2T 2easy DH 5Α, PCR CA PN 1, GeneBank A Y197555, 98%, 89%, 89%, 83%, 96% 81%, CA PN 1 EST CA PN 1, RNA, 2 [ ] nested touch2dow n PCR; EST ; CA PN 1 ; [] Q 343 [] A [ ] 167129387 (2005) 0220014205 PCR,, PCR ;,, M g 2+ PCR, Don [1 ] touch2dow n PCR, PCR ( calpain ) 2, Λ2calpain (CA PN 1) m 2calpain (CA PN 2), 2, 30 ku 2,, 80 ku 2 [2 ] CA PN 1 [3, 4 ], CA PN 1, [5 ] CA PN 1 EST [6, 7 ] 2, BTA 15 [8 ], BTA 29, CA PN 1 [9 ], CA PN 1 EST R T, nested touch2dow n PCR RNA CA PN 1 EST,, EST CA PN 1, CA PN 1 1 1. 1 TR Izo l T hermoscrip t TM R T 2PCR System Invitrogen ; LA T aq DNA S a lg T akar a ; pgem 2T 2easy T 4 DNA Xgal P rom ega ; E. coli DH 5Α Ξ [ ] 2004203216 [ ](03130) [ ] (1973- ),,,, E2m ail: caixin2323@ 126. com [ ] (1960- ),,,,, E2m ail: w ujp@gsau. edu. cn

2 : nested touch dow n PCR CA PN 1 EST 15 1. 2 1. 2. 1 C lastalw GeneBank CA PN 1 cdna, PCR ( 1) Cai3 (5 p rim er) : 5 2GAA GGA T GGCCGA GGA GT TCA TCA C23 ; Cai4 (3 p rim er) : 5 2GTCTCA CGCAAA CA T GGTCA GCT G23 ; Cai5 (5 p rim er) : 5 2CCT GA TCCGGA T GCGGAA CCCCT G23 ; Cai6 (3 p rim er) : 5 2GT GCTCCCCCGCCGCCA GGT GC23 1 PCR 5 3CA PN 1 ORF ( ) : Cai3 Cai4 2 132 bp; Cai5 Cai6 226 bp F ig. 1 Schem atic diagram of the nested PCR amp lification strategy fo r CA PN 1 EST of B os g runniens ORF of CA PN 1 containing 5 and 3 untranslated regions: the sequence betw een Cai3 and Cai4 is 2 132 bp; betw een Cai5 and Cai6 is 226 bp 1. 2. 2RNA TR Izo l RNA : 1 ml TR Izo l+ 50 100 m g, 15 30 5 m in0. 2 ml 4, 12 000 g 15 m in 0. 5 ml,, 20 m in, 15 30 10 m in 4, 12 000 g 15 m in 500 ml 70% RNA, 4, 12 000 g 5 m in RNA ( ) RNA SD S - 70 1. 2. 3 R T, nested PCR touch dow n PCR T hermoscrip t TM R T 2PCR System R T, cdna nested PCR: Cai3 Cai4 1 PCR, 25 ΛL, 10 PCR buffer 2. 5 ΛL, dn T P s 2. 5 ΛL, R T 1 ΛL, 0. 5 ΛL, LA T aq DNA 0. 2 ΛL, touch dow n PCR : 94 4 m in; 96 20 s, 58 1 m in, 72 90 s, 3 ; 96 20 s, 56 1 m in, 72 90 s, 3 ; 96 20 s, 54 1 m in, 72 90 s, 3 ; 96 20 s, 52 1 m in, 72 90 s, 3 ; 96 20 s, 50 1 m in, 72 90 s, 28 ; 72 7 m inpcr 1%, EB, 1 PCR 0. 5 ΛL, Cai3 Cai6, Cai5 Cai4 2, 1 PCR, 1 PCR, PCR, EB, 2 PCR 1 ΛL, Cai5 Cai6 3, 1 PCR, : 94 4 m in; 96 20 s, 50 1 m in, 72 90 s, 30 ; 72 7 m inpcr, EB, 1. 2. 4 PCR PCR silica pgem 2T 2easy, DH 5Α, LA 3 ml LA,, S a lg PCR 1. 2. 5 DNA AB I PR ISM 3100 Genetic A nalyzer CA PN 1 EST N CB I B lastn, C lu stalw 2 2. 1 R T, nested PCR touch dow n PCR CA PN 1 EST RNA OD 260gOD 280= 1. 56,, R T 2PCR R T 2 1 PCR, 1 PCR, Cai3 Cai6, Cai5 Cai4 2, 6 1, ( 2)Cai3 Cai6, Cai5 Cai6 3 PCR, 3, 1 270 bp, ; 2

16 () 33 540 1 600 bp, ( 3) 270 bp S a lg, ; Cai5 Cai6 PCR,, ( 4), DNA, 5 22 PCR M. DNA m arker (DL 2000) ; 1. Cai3 Cai6 ; 2. Cai5 Cai4 F ig. 2 R esults of the second2 round PCR am p lification M. M o lecular w eigh t m arker (DL 2000) ; 1. PCR p roducts using p rim ers Cai3 and Cai6; 2. PCR p roducts using p rim ers Cai5 and Cai4 33 PCR M. DNA m arker (DL 2000) ; 1. Cai5 Cai6 F ig. 3 R esults of the th ird2 round PCR am p lification M. M o lecular w eigh t m arker (DL 2000) ; 1. PCR p roducts using p rim ers Cai5 and Cai6 4 pgem 2T 2CA PN 1 PCR A. PCR ; B. S a lg ;M. DNA m arker (DL 2000) ; 1. pgem 2T 2CA PN 1 F ig. 4 PCR amp lification and restriction enzym e digestion of pgem 2T 2CA PN 1 recom binant p lasm id of B os g runniens A. PCR amp lification of the recom binant p lasm id; B. Restriction enzym e digestion of the recom binant p lasm id using S a lg ; M. DNA m arker (DL 2000) ; 1. pgem 2T 2CA PN 1 recom binant p lasm id of B os g runniens 5 CA PN 1 EST F ig. 5 N ucleo tide and deduced am ino acid sequence of EST fo r CA PN 1 of B. g runniens

2 : nested touch dow n PCR CA PN 1 EST 17 2. 2,, 224 bp CA PN 1 EST, GenBank A Y197555 N CB I B lastn,, 98%, 89%, 89%, 83%, 96% 81% ( 1 ) C lu stalw, CA PN 1 EST, 88%, 1 CA PN 1 EST T able 1 R esults of homo logical analysis of EST fo r CA PN 1 of B. g runniens w ith its countparts of o ther m am als Species Genebank A ccession N o. Sco re Expect value g% Identities B os g runn iens A F221129 504 e2140 98 O v is a ries A F071575 168 2e239 96 S us scrof a A F263610 400 e2109 93 H om o sap iens BC017200 313 7e283 89 M acaca f ascicu la ris A F284440 305 2e280 89 M us m uscu lus XM 192846 183 3e242 83 R a thus norveg icus NM 019152 147 9e233 81 3,,, 90 %, 1g6 [10 ],,, 3, Cai32Cai6, Cai52 Cai4 Cai52Cai6 nested PCR, touch2dow n PCR [1 ] PCR,, 2 58 50, RNA, RNA CA PN 1,, EST, nested touch2dow n PCR RNA, CA PN 1 EST, 98%, 96% 93%, 89%,, CA PN 1 CA PN 1, EST, CA PN 1, [ ] [ 1 ] Don R H, Cox P T,W ainw righ t B J, et al. Touch2dow n PCR to circum vent spurious p rim ing during gene amp lification[j ]. N ucleic A cid Research, 1991, 19 (14) : 4008. [ 2 ] C roal D E, D em artino G N. Calcium 2activated neutral p ro tease (calpain) system: structure, functure, and regulation [J ]. Physio logical Review s, 1991, 71 (3) : 813-847. [ 3 ] Suzuk i K, So rim ach i H. A novel aspect of calpain activiation[j ]. FEBS L ett, 1998, 433 (1-2) : 1-4. [ 4 ] A zam M, A drabi S S, Sah r K E, et al. D isrup tion of mouse m u2calpain gene reveals an essential ro le in p latalet function[j ]. M o l Cell B io l, 2001, 21 (6) : 2213-2220. [ 5 ] Koohm araie M, Babiker A S, M erkel R A, et al. Ro le of Ca 2+ 2dependent p ro teases and lyso som al enzym es in po stmo rtem changes in bovine skeletal m uscle[j ]. J Food Sci, 1988, 53: 1253.

18 () 33 [ 6 ] A ok i K, O hno S, Emo ri Y, et al. M o lecular cloning of the cdna fo r the large subunit of the h igh2ca 2+ 2requiring fo r hum an Ca 2+ 2 regulated neutral p ro tease[j ]. B iochem, 1988, 27: 8122-8128. [ 7 ] Po irier C, Poussard S, Faust D M, et al. M app ing, cloning, cdna sequence, and exp ression of the gene of the mouse m icromo lar calpain large subunit[j ]. M amm Genom e, 1998, 9: 388-389. [8 ] Keele J W, Shackelfo rd S D, Kappes M, et al. A region on bovine ch romo sone 15 influences beef longissim us enderness in steers [J ]. J A nim Sci, 1999, 77: 1364-1371. [ 9 ] Sm ith T P L, Casas E, Rexroad C E, et al. Bovine CA PN 1 m ap s to a region of BTA 29 containing a quatitative trait locus fo r m eat tenderness[j ]. J A nim Sci, 2000, 78: 2589-2594. [ 10 ]. [J ]., 1996, 26 (3) : 33. Iso lation of EST fo r CA PN 1 of B os g runn iens w ith nested and touch2dow n PCR CA I X in 1,W U J ian-p ing 2, XU M ing-xu 3, ZHANG L i-p ing 2, W ANG Hong-y ing 4,L IU Ya-nan 3 (1 Colleg e of L if e S ciences,n orthw est A & F U niversity, Y ang ling, S haanx i 712100, Ch ina; 2 Colleg e of A nim al S cience and T echnology, Gansu A g ricu ltu ral U niversity,l anz hou, Gansu 730070, Ch ina; 3 H um an D isease Genom ics Center, P ek ing U niversity,b eij ing 100083, Ch ina; 4 T he F irst M id d le S chool of Z hang x ian County, Z hang x ian, Gansu 748300, Ch ina) Abstract: CA PN 1 gene w as investigated as candidate gene affecting m eat tenderness. W ith the p rim ers designed acco rding to cdna s encoding CA PN 1 pub lished in GeneBank, R T, nested and touch2dow n PCR w ere u sed to iso late EST of to tal RNA s from m u scle of B os g runn iens. T he PCR p roduct w as ligated to pgem 2T 2easy vecto r, and then E. coli DH 5Α strain w as tran sfo rm ed. Po sitive clones w ere iden tified by restriction endonuclease, PCR and sequencing. T he partial sequence encoding CA PN 1 of B os g runn iens w as iden tified, the co rresponding EST s of B os tau rus, H om o sap iens, M acaca f ascicu la ris, M us m uscu lus, O v is a ries and R a ttus norveg icus shared 98%, 89%, 89%, 83%, 96% and 81% homo logy w ith that of B os g runn iens respectively. It indicated that the EST w as comparatively con servative betw een differen t species. Iso lation of the EST has m ade sub stan tial basis fo r fu rther research on CA PN 1 gene of B os g runn iens. T he resu lt of th is resea rch suggest s tha t the com b in ing u se of nested and touch2dow n PCR is an effect ive m ethod in EST iso lation even though to tal RNA s are partially degraded. Key words: nested and touch dow n PCR; EST iso la t ion; gene fo r la rge subun it of CA PN 1; B os g runn iens