classical pcr - ligation strategy is the most familiar one to clone for its convenience and efficiency . in long cds cloning , pcr and sequencing are the most challenging steps .
dna is not easily amplified from complementary dna ( cdna ) for many reasons , e.g. cdna may not be in good quality , primers are easier to miss - prime during long extending time , gc - rich region will stop dna polymerizing . on the other hand , fidelity is far from our satisfactory .
in addition , long fragment sequencing will be troublesome for both time and money cost on intermediated sequencing and primer synthesis .
that 's why large gene , in many cases , have to be interrupted into several domains to imitate the whole gene function 1 . to solve the problem ,
somebody will turn to golden gate ligation system . because the cutting sequences of the two enzymes are not restricted
, the overhanging 4 nucleotides could give at most 256 different patterns of cohesive ends .
this dramatically increases the choices for our consideration to assemble small fragments into a long one 5 - 7 .
however , if our target long cds contains bbsi or bsai restriction sites , golden - gate ligation system will not work well .
besides , this strategy requires elite skills on cohesive end selecting and designing , it will not be favored by many scientists even though it is widely used in talen tandem assembly 8 .
we found a strategy to clone large fragments of dna by dividing them to short pcr fragment cloning , followed by restriction digestion and ligation cloning 9 .
this strategy shows dominance in amplifying efficiency , it helps overcome pcr problem , and constructs very big clones without turning to complex reagents and technologies , like red / et system10 or tar system 11 , 12 .
this strategy shows potential in handling long cds clones , including introducing mutation , deleting and inserting domains . here
, we name it restriction - based multiple - fragment assembly strategy . a slight modification was added to make it more compatible to sequencing system and easier for us to handle random mutation .
a series of experiments were done to judge its efficiency and universality . in this modified strategy , instead of
pcr - amplifying the entire cds , we amplify short fragments , which are no longer than 1,500bp , spanning the whole cds . since sanger sequencing 's read length is 800bp , 1,500bp is very friendly to bi - directional sequencing strategy .
sequence - proved fragments were released by restriction digestion and assembled into our target vectors in a ligation system .
since the digestion and ligation sub - cloning does not introduce random mutation , and restriction sites were found in the sequence to be cloned , ligation will not break open reading frame ( orf ) . in this study ,
mago2 ( nm_153178.4 ) was submitted to nebcutter v2.0 ( http://nc2.neb.com/nebcutter2/ ) 13 for restriction analysis .
results showed mago2 contains an xhoi restriction site which breaks mago2 cds into 1.4 kb and 1.1 kb two fragments .
each interrupted fragments were pcr - amplified from n2a cdna using primers mago2f1 ( 5'-acg gat ccg cca cca tgt act cgg gag ccg gcc ccg ttc-3 ' ) , mago2r1 ( 5 ' -act tgc ata cac agg agt t-3 ' ) , mago2f2 ( 5'-agc gcc agt gta cag aag tc-3 ' ) mago2r2 ( 5'-acg aat tca gca aag tac atg gtg cgc ag-3 ' ) by kod - fx ( cat : kfx-101 ) .
pcr was processed 28 cycles on eppendorf thermos - cycler ( eppendorf ag 22331 hamburg ) with the denaturing temperature at 94 for 30seconds , annealing temperature at 58 for 30seconds , followed by extending temperature at 68 for 1min / kb .
pcr product was purified ( guangzhou dongsheng biotech ) and ligated ( fermentas # k1423 ) to pbluescript - ksii that had been digested with ecorv to give blunt end .
ligation product was transformed into top 10 competent e. coli . and plated onto lb agar plate supplemented with x - gal , iptg and ampicillin .
purified fragments were ligated to pcdna3.1-myc / his a that had been digested by bamhi and ecori in one ligation system .
this strategy to clone long dna fragment takes advantage of the dominance of short fragment pcr .
but , full - length pcr product showed smear at 500bp and 1,000bp ( figure .
after our analysis , hxrn1 cds would be divided into 4 short fragments ( a , b , c , d ) with their lengths at 0.7 kb , 1.5 kb , 1.4 kb , 1.4 kb adjoined by endonucleases aflii , hindiii , ecori .
at the same time we got ksii - cd . again , we repeated the processes above and got pcdna3.1-myc / his - hxrn1 ( figure .
hxrn1 full length pcr showed no band at all ; while fragments pcr shows clear and bright bands ( figure .
this means that short fragments are much more easily amplified and products are more accurate .
next , hxrn1 4 fragments were used to measure one round assembling efficiency of 2 fragments ( a / b ) , 3 fragments ( a / b / c ) and 4 fragments ( a / b / c / d ) .
after overnight growth , 20 clones of each plated were picked for colony pcr . in 2 fragments system ,
16 positive clones were checked , in 3 fragments system , 15 positive clones were checked , in 4 fragments system , only 6 positive clones were checked ( figure 3 ) .
results showed that the more fragments are tried in an assembly system ; the lower success rate is checked . to overcome the efficiency problem , we recommend two or more round of assembly , as it has been processed on pcdna3.1-hxrn1 . in order to know how universal this strategy is , we searched the entire human and mouse cds in ccds database 15 to see how many of them will benefit from our strategy .
sequences were checked in our python script ( supplementary material s1 ) if they were longer than 1,500bp , if 15 candidate restriction sites ( apai , bamhi , bglii , ecori , hindiii , kpni , ncoi , ndei , nhei , noti , saci , sali , sphi , xbai , xhoi ) could be found only once on a cds , and then if the distance between adherent restriction sites are no longer than 1,500nt .
our data showed 12118 in 29064 human cdss and 9479 in 23874 mouse cdss were longer than 1,500 ( supplementary material s2 ) .
further , 8304 in 12118 in human and 6678 in 9479 mouse meet our requirements ( supplementary material s3 ) .
sequences meet our requirement are good candidates for restriction - based multiple - fragment assembly strategy .
this data strongly suggests our strategy is potential to solve most long cds cloning problem .
furthermore , we can increase the number by referring more restriction sites or turning to other ligation free system 16 . in this study , first , we discuss multiple - fragment assembly strategy 's dominance in pcr amplifying efficiency by amplifying short fragment rather than long one .
fourth , we programmed a script to test its universality in the entire cds , and find a great number ( ~70% ) of long cds problem will benefit from it .
 long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation . here 
 we discuss restriction - based multiple - fragment assembly strategy 's advantages and limitations . in this strategy , rather than pcr amplifying the entire coding sequence ( cds ) at one time , we amplified and sequenced smaller fragments which are shorter than 1.5 kb spanning the cds . 
 after that , the sequence - proved fragments were assembled by digestion - ligation cloning to the target vector . 
 we test its universality in our script programmed in python . 
 our data shows that , among the entire human and mouse cds , at least 70% of long cds cloning will benefit from this strategy . 