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208 JouRnAl oF BiomoleculAR Techniques, Volume 17, issue 3, July 2006

(c) Containing hairpin structures. Technically these struc-tures consist of two inverted repeats, separated by atleast three nucleotides. They warrant classification in aseparate category due to the increasing importance of si/shRNA-based research that quite often requires sequenc-ing through such structures.7,913

Strong hairpin structures are also an integral part of

some vectors, e.g., the pDONR/pDEST series from Invi-trogen (www.invitrogen.com) or the inverted terminalrepeat sequences in adeno-associated viruses.

(d) Containing long homopolymer stretches. Most typical arepoly-A/T tails resulting from constructs obtained throughreverse-transcriptase (RT) PCR amplification of mRNAs.

The length of such tails varies from around 20 to over 100A/Ts. The poly-G/C stretches not only contribute to theoverall GC richness but also may form strong hairpinsand other complex secondary structures.

(e) Containing sequence motifs causing band compressions.14These are predominantly 5-YGN12AR motifs, where Y

and R are pyrimidine and purine residues, respectively,and N can be any base.

One of the complicating factors in sequencing manydifferent categories of difficult templates is the increas-ingly apparent realization that there may not be a one-method-fits-all solution and that each category requires aseparate approach (or even set of approaches). A numberof papers describe modified sequencing protocols. How-ever, they appear to apply only to specific types of diffi-cult templates at best. 47,1519

On the other hand, a single approach that incorpo-

rates a 5-min heat-denaturation step (with or withoutsome other additives) was successfully applied to manydifferent categories of difficult templates.7,13,20

In this paper, we review both general and specificapproaches to sequencing many different kinds of dif-ficult templates, starting with the description of theheat-denaturation step and its impact on the quality ofsequencing data.

MATERIALs And METhods

All materials and methods presented in this review wereextensively described in earlier publications7,13,20,27,29,31

and will be not repeated here unless there is a notabledeviation. However, for convenience, we will definestandard and modified sequencing protocols. Standardsequencing protocolrefers to a basic protocol recommendedby Applied Biosystems technical literature, as described,for example, in reference 3, with the exception that thefinal volume is 10 L. Briefly, DNA, primer, water, anddye terminator mix are combined, and cycled 25 times:96C/10 sec, 50C/5 sec, 60C/4 min. In the modified

sequencing protocol, we combine DNA, primer, and10 mM Tris (pH 8.0), heat-denature samples for 5 min at98C, and then add dye-terminator mix. If additives areused, they are included in the heat-denaturation step.

ConTRoLLEd hEAT dEnATuRATIon of pLAsMIds

In the early days, DNA sequencing was dominated by non-

thermostable polymerases, primarily the T7-based Seque-nase,2123 and to sequence any double-stranded template,the first step needed to be strand separation for the effi-cient annealing of the primer. There were many protocolsdesigned for strand separation, all including heating (22Cto 100C) in the presence or absence of 0.1 to 0.3 N NaOHfollowed by neutralization, precipitation, and re-suspen-sion in the desired solution.2427 These protocols weretime-consuming, cumbersome, and insufficiently reliablefor routine use. Instead, a simple heat-denaturation step

was suggested in low-salt buffers and at elevated tempera-tures, to convert supercoiled plasmid DNA efficiently to

a single-stranded form amenable for sequencing.20 Thedenaturation can be carried out in water, but it occasion-ally produces additional bands that effectively reduce theamount of template available for sequencing (J. Kieleczawa,unpublished observation). The time needed to convertDNA effectively from supercoiled form to single-stranded(ss) form depends on the size of the plasmid; the biggerthe plasmid, the shorter time needed for this conversion.It takes 7.5 min (10 mM Tris-Cl, pH 8.0 buffer, 98C) toconvert 75% of pGem3zf (3.2 kbp) from supercoiled toss form. (The 75% conversion level is selected to avoid

potential degradation of DNA.) For any plasmid biggerthan pGem3zf, one needs to subtract 1 min per multipleof 2.5 kbp from 7.5 min to achieve a similar level of dena-turation. In fact, there is a linear relationship between sizeof a plasmid, at least in the 3- to 20-kbp size range, andtime needed for efficient conversion to a form suitablefor sequencing.20 As a rule of thumb, the time neededto achieve a similar level of denaturation in water shouldbe halved compared to the time in 10 mM Tris-Cl buf-fer. Furthermore, the linear relationship between the sizeof the plasmid and the time needed for conversion holdstrue only for plasmids that do not contain any difficult

regions. For example, templates rich in GC or with CTTrequire 30 and 20 min, respectively, for 75% conversion(J. Kieleczawa, unpublished observation). Surprisingly,plasmids with long poly-A/T tracts (7080 bases) alsorequire up to 20 min for effective conversion to ss formunder similar conditions. In the presence of 2 mM MgCl2(the final concentration of Mg ions under optimal cyclesequencing conditions using ABIs dye-terminator chem-istry3), there is no, or very little, conversion of supercoiled

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FunDAmenTAls oF sequencinG oF DiFFiculT TemPlATes

JouRnAl oF BiomoleculAR Techniques, Volume 17, issue 3, July 2006 209

to ss form either during prolonged heat denaturation at98C or during cycle sequencing (Figure 1). It appears that

the primary part that is transformed to a form amenableto sequencing is the nicked form; hence, one needs to usemore DNA than necessary to compensate for the partialconversion. Table 1 shows the sequence data for differ-ent NaOH-induced plasmid denaturation protocols andthe denaturation conditions recommended in this work.Based on the data presented in references 7, 20, and 28,there are the following advantages to incorporation of theheat denaturation step into a sequencing protocol:

(a) The conversion of supercoiled DNA to ss formand destruction of any residual DNAse activity is veryeffective.

(b) The amount of DNA needed for optimal resultsis three- to fivefold lower than that recommended by astandard ABI protocol.

(c) At a given amount of DNA, the fluorescent signalstrength is approximately threefold higher than that ofsamples without a heat-denaturation step (which is directlyrelated to point a above).

(d) Read length (Q 20) is increased by 72.3 20(~10% more) for standard (nondifficult) templates.

(e) Very effective when sequencing many types of dif-ficult templates. In all cases, clearer data are generated

with this step, and in 7/22 cases the heat-denaturationmethod succeeded in generating readable data where stan-dard methods failed (see Table 2). [AU: Table 1 has notbeen cited. Please indicate where it belongs.]

(f) First correctly called base is 35 nucleotides closerto the 3 end of the primer (for M13 primer with pGem3zfDNA).

(g) Fewer sequencing errors (4.4 errors/1000 bases)are detected than with a standard protocol.

The kinet ics of reversal from ss form back to super-coil-like form is extremely slow (measured in days) both inthe presence or absence of 2 mM MgCl2 and at tempera-

tures ranging from 0 (ice) to 22C (Kieleczawa, unpub-lished observation). So, even if the assembly of sequencingreactions takes a few hours, as it could in a factory-likesequencing pipeline, there is no need to take any specialprecautions after the initial heat denaturation (performed

with DNA, primer, low-salt buffer, or water) and beforethe addition of dye-terminator mix. Furthermore, aspointed out by Kieleczawa and Wu,29 the dye-terminatormix can be stored for several days at room temperature

fIguRE 1

Datrat f pG3f dr dffrt dt.Tw drd agra f pG3f wa datrd gat-datrat dt drbd t papr ad vra pbd papr. (1) tr pG3f;

(2) 2.5 at-datrat (hD); (3) 7.5 hD; (4) 15 hD (DnA a 14 wa 10 m Tr-c, 0.01m eDTA (ph 8.0) = Te. T DnA a 514 wa a Te bt ad addta pt): (5) tpr f 2 m mgc2, 7.5 hD; (6) t pr f BgD tratr v3.0/4X fa dt wt mgc2at 2 m, 7.5 hD; (7) t pr f 2 m mgc2 fwd b 40 g [(10 /96c)(5 /50c)(2 /60c)]; (8) a a 6 bt f wd b g a a 7; (9) 7.5 hD, add 2 m mgc2 fwdb g a a 7; (10) 7.5 hD, add d-tratr a a 6, fwd b g a a 7; (11) tpr f 0.1 n naoh batd at r tpratr fr 5 , trad wt 0.1 n hc;25 (12) t pr- f 0.2 n naoh batd at r tpratr fr 5 , trad wt 0.2 n hc;26 (13) t prf 0.2 n naoh batd at 85c fr 5 , trad wt 0.2 n hc;26 (14) t pr f 0.1 n naohbatd fr 3 at 100c, trad wt 0.1 n hc.27

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(or even at 37C) with no adverse influence on the qual-ity of the resulting sequences. Hence, all assembly andpre-treatment steps can be safely performed at room tem-perature, with the only necessary precaution being controlof evaporation of the sequencing mix to avoid excessiveimbalance in the dNTPs and Mg ion concentrations.

See Figures 2, 3, and 4 for examples of the effect ofheat denaturation on the sequence quality.

ExAMpLEs of sEquEnCIng of

vARIous dIffICuLT TEMpLATEs

In this section we briefly describe possible modificationsthat present the best chance(s) to sequence through spe-cific types of difficult templates.

(a) Sequencing of GC-rich templates. This category of tem-plates is arguably the most commonly encountered in atypical sequencing laboratory (DSRG survey, 2005 data,unpublished). Most often, the solution of choice is toadd DMSO to the final concentration of 2.55%, andit seems to be effective in templates with up to 6072%

GC content.4 6 On the other hand, the inclusion of a 5-min heat-denaturation step is even more effective7 and,of course, does not require the addition of DMSO. In ourDNA sequencing core laboratory, we have developed pro-tocols (built into our laboratory information managementsystemLIMS) that automatically switch between vari-ous chemistries depending on the GC content of a partic-ular region in a template. When GC content is 70%, justheat denaturation alone is sufficient to sequence a major-

T A b L E 1

Comparison of Read Lengths and Signal Strengths for Varying DNA Concentrations and Different Denaturation Conditions

Datratcdt

T Wrk/h20a T Wrk/Teb

Rf. 25 Rf. 26c Rf. 26d Rf. 27-hD +hD -hD +hD

25 g

DnA

Rl

q 20

489.587.3 744.353.8 488.084.7 724.771.0 268.662.1 284.1124.5 223.692.2 0

ss 13.03.7 32.16.1 12.32.5 27.26.0 8.82.2 10.63.8 8.62.0 5.80.5

50 gDnA

Rlq 20

585.4177.7 764.244.1 625.4106.0 784.959.1 577.444.3 409.959.0 436.793.0 339.090.9

ss 15.65.2 47.08.6 16.93.4 45.819.8 12.62.5 10.52.2 9.41.8 7.61.2

200 gDnA

Rlq 20

837.725.1 843.622.2 724.271.5 866.429.0 792.039.8 663.3113.7 621.276.8 712.841.4

ss 59.89.8 119.214.1 41.77.4 123.320.1 65.12.2 43.19.7 29.88.2 26.15.9

Varg at f pG3f pad DnA (data ar t avrag f fr ap fr a dt) wr prtratd dffrtbfr bjtg t t g. Prd g rat wr r a ABi3100 ppd wt a 80- ap-ar arra a rdd b t afatrr. T data wr vaatd tr f q 20 rad gt (Rl) ad ga trgt (ss

frt t a prdd b a ABi trt).aDnA wa rpdd watr ad d g tadard ABi prt (-hD = at datrat), r ap wr atdatrd (+hD) fr 2.5 at 98c bfr g.

bDnA wa rpdd 10 m Tr/0.01 m eDTA (ph 8.0) ad d g tadard ABi prt (-hD), r ap wrat datrd fr 7.5 at 98c bfr g.

cDnA wa datrd at r tpratr (22c) fr 5 t pr f 0.2 n naoh. Fwg t tratt, a a v- f 0.2 n hc wa addd t tra t t. T xtr wr pptd wt 40 l f watr, 6 l f 3 m datat (ph 6.0), ad 160 l 95% ta, ad wr trfgd fr 30 at 3400g. spratat wr dardd, ad prptatwr wad tw wt 200 l f 70% ta. Fa, ap wr drd fr 15 at 65 c ad rpdd 6 l f watr,1 l 5 m m13 rvr prr, ad 3 l f twfd dtd BgD tratr v3.0, ad trprd a drbd rfr20.

dA drbd dr ftt c, bt datrat wa at 85c.

T datrat dt fr rfr 25: DnA wa datrd fr 5 at r tpratr t pr f 0.1 n naohad trad wt 0.1 n hc. Rag tp wr a drbd abv.

T datrat dt fr rfr 27: DnA wa datrd fr 3 at 100c t pr f 0.1 n naoh fwd btraat wt 0.1 n hc. Rag tp wr a drbd abv.

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ity of difficult regions. When the GC content is between70 and 80%, we add reagent A (from a panel of seven Rxreagents sold by Invitrogen, Inc., Carlsbad, CA) or 1 Mbetaine (purchased from Sigma-Aldrich, St. Louis, MO).For templates with GC content from 80 to 90%, we usedGTP V3.0 chemistry in conjunction with reagent A or C.

It is worth remembering that dGTP Big Dye occasionallyproduces band compression and needs to be correctedusing standard BigDye chemistry.13 Above 90% GC con-tent, we (through our LIMS) apply the two-step processthat includes PCR amplification of the fragment in ques-tion, using 7-deaza-dGTP instead of dGTP, followed by

T A b L E 2

Sequencing Difficult Templates Using Various Protocols

DnATp

sg mtd

mtd n.

1 2 3 4 5 6 7 8

DnA caratrt -hD +hD -hD +hD +hD +hD +hD +hD

1 60% Gc 834 868 818 862 895 AcG 884 Bt 511 2802 63% Gc 208 572 nT 121 673 c 635 Gc 552 549

3 66% Gc 538 847 867 854 894 AG 858 Bt 728 635

4 67% Gc 0 989 nT 222 1087 c 1025 Gc 177 945

5 75% Gc 0 660 nT 960 974 F 940 Bt 747 713

6 80% Gc 661 920 914 920 940 AcG 933 Bt 842 848

7 86% Gc 0 504 nT 928 967 c 988 Gc 895 675

8 94% Gc 0 277 nT 270 304 A 302 Bt 318 420

9 cT rpat 569 886 107 618 950 c 815 Bt 845 927

10 GA rpat 363 761 168 416 900 AcG 825 Bt 580 765

11 cTT/TTTccc rpat 222 277 nT 193 914 cBD 898 Bt 477 nT

12 Tcc/Gcc rpat 0 830 nT 853 929 c 893 Bt 855 nT

13 cTT rpat 401 431 nT 411 540 Bce 466 Bt 455 426

14 ccccnn/TTcc rpat 390 609 503 805 1000 AFG 1000 Bt 954 890

15 GGGGnn/AAGG rpat 378 991 873 1008 1011 AcG 995 Bt 882 886

16 A rpat 0 118 nT 107 262 BDe 258 Gc 820 nT

17 19 G pr 706 743 809 834 860 Ac nT 712 nT

18 19 c pr 487 537 403 409 856 Ac nT 471 nT

19 23 G pr 283 404 407 418 606 A nT 605 nT

20 28 ba iTR arp* 410 439 nT nT 462 c nT nT nT

21 RnA pad frwardprr

0 590 nT 601 710 AcD 885 Bt 811 811

22 RnA pad rvrprr

566 820 nT 750 806 cD 806 Gc 963 955

Twt-tw dffrt dff t DnA wr d g a vart f prt. mtd 1 vat t ttadard ABi prt.3 mtd 2 vat t prt drbd rfr 7, 13, 20 (d 5 at-datrat tp). mtd 3 k prt 1 bt t pr f 5% Dmso. mtd 4 k prt 2, btDmso wa dd drg at-datrat tp. mtd 5 k prt 2, bt f v ragt frivtrg (carbad, cA) ar Rx t wa prt drg datrat tp ( data fr t -gt rad ar rprtd r). mtd 6 k td 2, bt dd tr addtv (Gc t fr BDB, sa J, cA; Bta wa fr sga-Adr, st. l, mo). mtd 7 wa k td 2, bt BgDV3.1 wa btttd wt dGTP V3.0. mtd 8 wa k td 2, bt BgD V3.1 wa btttd wt 4:1 (v/v)x f BgD V3.1 ad dGTP V3.0 a ggtd b G. Gr (pra at). A data ar xprd q 20 va a drbd arr.7

*Fr t tw tpat, t rrt avaab td f radg trg t app a tw-tp prt a

drbd rfr 7.

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then 19 bases, the two-step approach described above mayneed to be used. A variety of protocols are recommendedfor sequencing through poly-A or -T tails. Poly-A/T B(V) N primers (where B is not A and V is not T: N is anybase) are commonly used and are sometimes effective.19

Another approach is to use a primer that spans the bound-

ary of a unique sequence and the poly-A/T tail,18 but wehave found that this is not universally applicable. Ourpreliminary observations suggest that successful readingthrough long poly-A/T stretches (especial ly >50) dependson the sequence that immediately follows such stretches;difficult regions immediately following an A/T tail willmake a clean sequence read harder, while it is possible toget readable A/T sequence if the downstream region isnot difficult. One effective way, although not so straight-

forward, to deal with such situations is to get reads fromboth ends, often using a new primer in reverse orientation.It follows that quite often one will not have full double-stranded coverage of such stretches.

IsoLATIon of dIffICuLT TEMpLATEs wITh

vARIous CoMMERCIAL kITs: EffECT on

sEquEnCE quALITy

It is well established that the most important factor influ-encing good sequencing data is the quality of the DNApreparation, assuming the amount of DNA is sufficient.

As the chemistry and sample treatment vary among com-mercial kits, it is reasonable to expect that there will bedifferences in quality of sequence data obtained whenthe same DNA is prepared using different kits.30 In our

fIguRE 3

cratgra f Gc-r tpat (abt 72%)d g a tadard (A) ad dfd (b)prt. T solid bardat dta rg

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J. KieleczAWA


studies31 we prepared twelve different difficult DNAsusing six conventional DNA preparation methods anda Templiphi-based protocol to evaluate whether there isany advantage using one kit over another. Two well-estab-lished commercial DNA kits (from EdgeBiosystems andQiagen) seem to be most consistent in producing high-quality DNAs leading to the longest reads,31 as shownin Table 3. The Templiphi produces branched and some-

what unstructured molecules32,33 that, we were expecting,would be easier to read through, but unfortunately thisturned out to be incorrect. The technical literature from

Amersham34 (currently part of GE Healthcare) claims thatcertain types of difficult templates, prepared using Tem-

pliphi, are easier to sequence compared to those isolatedwith conventional methods. However, this claim needs tobe independently verified.

ThE wEb As A REsouRCE foR InfoRMATIon

on sEquEnCIng of dIffICuLT TEMpLATEs

In the era of Yahoo and Google, it is only natural that

one would be tempted to scan the Web for information/advice regarding sequencing of difficult templates. Typ-

ing, e.g., diff icult DNA templates into Googles searchengine results in about 341,000 hits (211,000 in Yahoossearch engine), which leads to about 1000 sites as of

January 2006. Rarely, though, does one get any advicebeyond DMSO or glycerol for GC-rich samples, orsome unspecified proprietary treatment in a few com-mercial sites. Quite unusual is also the fact that almostall sites cite the same/similar amounts of DNA neededfor optimal read length, concentrations of interferingagents, etc., without providing references to any pub-lished or unpublished data. An example of such unsub-stantiated advice is the recommended amount of PCRfragment needed for optimal read length: use 10 ng of

DNA/100 bases. In our experiments28

we demonstratethat even for a PCR fragment of 1250 base pairs, 1 ng ofDNA is fully sufficient for optimal read length. In fact,

we carr ied out simi lar experiments on PCR fragments ofabout 500 and 900 bases (with the amount of DNA/reac-tion varied from 0.1 ng to over 500 ng) and the resultsdid not support the 10 g/100 bases claim ( J. Kieleczawa,unpublished observation); in all cases, just 1 ng of DNAgave the optimal read length.

fIguRE 4

A xap f a tpat wt 59-ba GA -rpat trt d g a tadard (A) ad dfd (b)prt. T vw rprt at r a dpad b sr prgra. T light blue colordatba wt gr q at ad darker blue colordat ba wt wr q va. T black barw trg wt t a .

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Therefore, a reader (PC user) seeking fast Web adviceconcerning any aspect of DNA sequencing needs to behighly skeptical and selective, as the advice one gets maynot necessarily be helpful (although in most cases it willnot hurt either).

suMMARy

In this paper, we have reviewed past and current methodsto sequence through many types of difficult templates.

Although a number of solutions were suggested over thelast few years, they all seem to be quite specific to a par-ticular type of diff icult template. The possible exception isthe heat-denaturation modification, which appears to bemore broadly applicable for several different difficult tem-plates and in combination with several additives gives thebest chance to obtain good-quality data. Data presented

in Table 2 show that for 32% of difficult templates, add-ing just a heat-denaturation step was the only possible wayto obtain any sequencing data. For 22% of templates, theimprovement was on the order of 510% in read length.Increase of 1050% in read length was evident for another14% of templates, and for the remaining 32% the increasein read length was on the order of 50175%.It is apparentthat more experiments need to be carried out to developmore general rules, and it is entirely possible that, in the

interest of time, any given very difficult template mayneed to be sequenced using a few different chemistries inparallel. One of the potential venues to speed the devel-opment of general rules for sequencing of difficult tem-plates is to organize a bank of well-characterized DNAsand involve the broad community in applying a range oftechnologies. The model of such community effort is wellestablishede.g., the DNA Sequencing Research Group(DSRG) conducted a study on the effect of DMSO onsequencing of some GC-rich templates.5,6 In collaboration

with bioinformaticians, who now have extensive compu-tational tools, it should be possible to look for a moredetailed correlation between sequence patterns, especiallyright before and after difficult regions, and the success ofa given chemistry. It is also conceivable that one of the bigsequencing centers, NIH or some other entity, will estab-

lish a special unit that wil l investigate in greater detail thesequencing of all types of diff icult templates, and the art

wil l become science.As we approach an era of $1000-a-genome sequenc-

ing, one still-unanswered question is how these new tech-nologies will deal with more complex regions. It is pos-sible thatdifficult regions will not present any challenges,as most of these methods rely on assembly of very shortoverlapping fragments. Hopefully, researchers who are

T A b L E 3

The Performance of Six Classical Plasmid Preparation Methods

DnA prparattd

DnA sg mtd n.

mtd 1 mtd 2 mtd 3 mtd 4

n. % n. % n. % n. %

edgBt 7 58.3 4 33.3 4 33.3 5 38.5

eppdrf 0 0 0 0 0 0 0 0

marg 2 16.7 0 0 0 0 1 7.7

Prga 0 0 0 0 0 0 0 0

sga 0 0 0 0 5 41.7 4 30.8

qag 3 25.0 8 67.3 3 25.0 3 23.0

Twv dffrt df ft tpat wr prpard g x ra kt. Data w wa t (n ad %) gv td prdd DnA tat rtd gt rad (ard

wt q 20 va) g fr g td. mtd #1 vat t a tadard ABi-k prt. mtd #2 dd at datrat . mtd #3 ad 4 wr t tw bttd fr a gv DnA tpat ad dd at datrat ad f addtv (rfr 7 fr dta). T twv tpat d t xprt a bt f tpatw Tab 2.

expaat fr t tab. n. = br f t t td gav t bt rt.

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directly involved in the development of new technologieswil l be able to answer such questions relatively soon.

ACknowLEdgMEnTs

I wish to thank Dr. Laird Bloom of Wyeth Research for criticalreview of this manuscript and many valuable suggestions. The sup-port and encouragement by the management of the Biological Tech-nologies department of Wyeth Research is also greatly appreciated.I would like to thank Jones and Bartlett Publishers, Sudbury MA,for permission to reprint table 1-1 from (J. Kieleczawa, ed) DNASequencing: Optimizing the Process and Analysis, 2005, and tables 1-1 and1-4 from ( J. Kieleczawa, ed) DNA Sequencing: Optimizing the Prepara-tion and Cleanup, 2006.

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2. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-ter-minating inhibitors. Proc Natl Acad Sci USA 1977;74:54635467.

3. BigDye Terminator v3.1 Cycle Sequencing Kit. Protocol. 2002.Part number 4337035 Rev. A. Applied Biosystems. Foster City,CA.

4. Adams PS, Dolejsi MK, Hardin S, Mische S, Nanthakamur B,Riethman H, et. al. DNA sequencing of a moderately difficulttemplate: Evaluation of the results from a Thermus thermophilusunknown test sample. BioTechniques1996;21:678.

5. Adams PS, Dolejsi MK, Grill s G, Hardin S, McMinimy DL,Rush J, et al. Effects of DMSO, thermocycling and editing on a

T A b L E 4

Comparison of Read Lengths between DNAs Prepared Using Either Templiphi or

Qiagen/Marligen Methods

DnAn. caratrt

Prparatmtd

DnA sg mtd n.

1 2 3

1 ccT/TTTccc TpP 340 515 594

qag 375 495 647

2 Tcc/Gcc TpP 635 640 626

qag 599 659 642

3 cTT/cccT/ccTT TpP 0 0 0

marg 337 458 454

4 A-rpat TpP 128 147 292

qag 214 240 259

5 90% Gc TpP 111 278 285

marg 0 292 327

6 88% Gc TpP 143 165 181

qag 313 502 587

7 75% Gc TpP 148 610 567

qag 565 642 600

8 63% Gc TpP 524 591 596

qag 556 575 596

9 63% Gc +trg arp

TpP

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template with 72% GC rich area: Results from the 2nd AnnualABRF sequencing survey demonstrate that edit ing is the majorfactor in improving sequencing accuracy. Ninth InternationalGenome Sequencing and Analysis Conference. Microb CompGenomics1997;2:198.

6. Adams PS, Dolejsi MK, Grill s G, McMinimy D, Morrison P,Rush J, et al. An analysis of techniques used to improve theaccuracy of automated DNA sequencing of a G C-rich tem-plate: Results from the 2nd Annual ABRF DNA Sequence

Research Group study. Available at: http://www.abrf.org;search for 2nd ABRF DNA Sequence Research Group Study.1999.

7. Kieleczawa J. Sequencing of difficult DNA templates. In Kielec-zawa J (ed), DNA Sequencing: Optimizing the Process and Analysis.

Jones and Bartlett Publishers, Sudbury MA, 2005, pp. 2734.8. Zhao X, Haqqi T, Yadav SP. Sequencing telomeric DNA tem-

plates with short tandem repeats using dye terminator cyclesequencing.J Biomol Tech2000;11:111121.

9. Shar p PA. RNAi and double-strand RNA. Genes and Dev.1999:13:139141.

10. Hammond SM, Caudy AA, Hannon GJ. Posttranslational genesilencing by double-stranded RNA.Nat Rev Genet2001:2:110119.

11. Brummelkamp TR, Bernards R, Agami R. A system for stableexpression of short interfering RNAs in mammal ian cells. Science

2002:296:550553.12. Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of genefunction in somatic mammalian cells using small interferingRNAs.Methods2002;26:199213.

13. K ieleczawa J. Simple modif ications of the standa rd DNAsequencing protocol allow for sequencing through siRNA hair-pins and other repeats.J Biomol Tech2005;16:220223.

14. Yamakawa H, Nakajima D, Ohara O. Identif ication of sequencemotifs causing band compressions on human cDNA sequencing.DNA Res1996;3:8186.

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