(12) United States Patent (10) Patent No.: US 7,354,708 B2 Hall Et Al

USOO7354708B2

(12) United States Patent (10) Patent No.: US 7,354,708 B2 Hall et al. (45) Date of Patent: Apr. 8, 2008

(54) INVASION ASSAYS 5,030,557. A 7/1991 Hogan et al...... 435/6 5, 108,892 A 4, 1992 Burke et al...... 435/6 (75) Inventors: Jeff G. Hall, Madison, WI (US); Victor 5,118,605 A 6/1992 Urdea ...... 435/6 I. Lyamichev, Madison, WI (US); 5,144,019 A 9, 1992 ROSS ... 536/27 Andrea L. Mast, Madison, WI (US); 5,210,015 A 5/1993 Gelfand et al...... 435/6 Mary Ann D. Brow, Madison, WI (US) 5,270,184 A * 12/1993 Walker et al...... 435,912 5,380,833. A 1/1995 Urdea 536,221 (73) Assignee: Third Wave Technologies, Inc., 5,401,830 A * 3/1995 McKenzie ...... 530,371 Madison, WI (US) 5,403,711 A 4/1995 Walder et al...... 435/6 5,407,795 A 4/1995 Kolberg et al...... 435/5 (*) Notice: Subject to any disclaimer, the term of this 5,422,253 A 6/1995 Dahlberg et al...... 435/91.53 patent is extended or adjusted under 35 5,427,930 A 6/1995 Birkenmeyer et al. ... 435/91.52 U.S.C. 154(b) by 90 days. 5,487.972 A 1/1996 Geland et al...... 435/6 5,494,810 A 2/1996 Barany et al...... 435/91.52 (21) Appl. No.: 10/033,297 5,541,311 A 7/1996 Dahlberg et al...... 536,23.7 5,545,729 A 8/1996 Goodchild et al...... 536,245 (22) Filed: Nov. 2, 2001 5,601,976 A 2, 1997 Yamane et al...... 435/6 (65) Prior Publication Data US 2002/0187486 A1 Dec. 12, 2002 (Continued) Related U.S. Application Data FOREIGN PATENT DOCUMENTS (63) Continuation of application No. 09/350,597, filed on EP O 411 18.6 A1 2, 1991 Jul. 9, 1999, now Pat. No. 6,458,535, which is a continuation of application No. 08/823,516, filed on Mar. 24, 1997, now Pat. No. 5,994,069, which is a (Continued) continuation-in-part of application No. 08/759,038, OTHER PUBLICATIONS filed on Dec. 2, 1996, now Pat. No. 6,090,543, which is a continuation-in-part of application No. 08/756, Abrams et al., “Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel 386, filed on Nov. 26, 1996, now Pat. No. 5,985,557, Electrophoresis and a GC Clamp.” Genomics 7:463-475 (1990). which is a continuation-in-part of application No. Akhmetzjanov and Vakhitov, “Molecular cloning and nucleotide 08/682,853, filed on Jul. 12, 1996, now Pat. No. sequence of the DNA polymerase gene from Thermus flavus, "Nucl. 6,001,567, which is a continuation-in-part of appli Acids Res. 20:5839 (1992). cation No. 08/599,491, filed on Jan. 24, 1996, now Altamirano et al., “Identification of Hepatitis C Virus Genotypes Pat. No. 5,846,717. among Hospitalized Patients in British Columbia, Canada,” J. Infect. Dis. 171:1034-1038 (1995). (51) Int. Cl. CI2O I/68 (2006.01) (Continued) CI2P 19/34 (2006.01) C7H 2L/02 (2006.01) Primary Examiner Frank Lu C7H 2L/04 (2006.01) (74) Attorney, Agent, or Firm—Casimir Jones, S.C. C7H 2L/00 (2006.01) (57) ABSTRACT (52) U.S. Cl...... 435/6: 435/91.1536/23.1; 536/24.3: 536/24.33: 536/25.3: 536/25.32 (58) Field of Classification Search ...... 435/6, The present invention relates to means for the detection and 435/91. 1, 183: 436/94: 536/23.1, 24.3, characterization of nucleic acid sequences, as well as varia 536/24.33, 25.3, 25.33 tions in nucleic acid sequences. The present invention also See application file for complete search history. relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid (56) References Cited cleavage structure in a site-specific manner. The structure specific nuclease activity of a variety of enzymes is used to U.S. PATENT DOCUMENTS cleave the target-dependent cleavage structure, thereby indi 4,511,502 A 4, 1985 Builder et al...... 260.112 cating the presence of specific nucleic acid sequences or 4,511,503 A 4, 1985 Olson et al...... 260.112 specific variations thereof. The present invention further 4,512.922 A 4, 1985 Jones et al...... 260.112 relates to methods and devices for the separation of nucleic 4,518,526 A 5, 1985 Olson ...... 260.112 acid molecules based on charge. The present invention also 4,683,194 A 7/1987 Saiki et al...... 435/6 provides methods for the detection of non-target cleavage 4,683,195 A * 7/1987 Mullis et al...... 435/6 4,683.202 A 7/1987 Mullis ...... 435/91 products via the formation of a complete and activated 4,775,619 A 10/1988 Urdea ...... 435/6 protein binding region. The invention further provides sen 4,818,680 A 4, 1989 Collins et al...... 435/6 sitive and specific methods for the detection of human 4,876, 187 A 10/1989 Duck et al...... 435/6 cytomegalovirus nucleic acid in a sample. 4,994,368 A 2, 1991 Goodman et al...... 435/6 5,011,769 A 4, 1991 Duck et al...... 435/6 23 Claims, 128 Drawing Sheets US 7,354,708 B2 Page 2

U.S. PATENT DOCUMENTS Brow et al., “Differentiation of Bacterial 16S rRNA Genes and Intergenic Regions and Mycobacterium tuberculosis kato Genes by 5,614.402 A 3/1997 Dahlberg et al...... 435/199 Structure-Specific Endonuclease Cleavage.” J. of Clin. Micro. 5,660,988 A 8, 1997 Duck et al...... 435/6 34:3129-3137 (1996). 5,691,142 A 11/1997 Dahlberg et al. . ... 435/6 Carballeira et al., “Purification of a Thermostable DNA Polymerase 5,698,400 A 12, 1997 Cotton et al...... 435/6 from Thermus thermophilus HB8, Useful in the Polymerase Chain 5,719,028 A 2/1998 Dahlberg et al...... 435/6 Reaction.” Biotechniques 9:276-281 (1990). 5,783,392 A 7, 1998 Seibl et al...... 435/6 Ceska et al., “A helical arch allowing single-stranded DNA to thread 5,792,614 A 8, 1998 Western et al...... 435/6 through T5 5'-exonuclease.” Nature 382:90-93 (1996). 5,795,763 A 8/1998 Dahlberg et al...... 435/194 Ceska et al., “Structure-specific DNA cleavage by 5' nucleases.” 5,830,664 A 11/1998 Rosemeyer et al. . ... 435/6 TIPS 23 (1998). 5,837,450 A 11/1998 Dahlberg et al...... 435/6 Copley and Boot, “Exonuclease Cycling Assay: An Amplified 5,843,654 A 12/1998 Heisler et al...... 435/6 Assay for the Detection of Specific DNA Sequences.” 5,843,669 A 12, 1998 Kaiser et al...... 435/6 BioTechniques 13:888-891 (1992). 5,846,717 A 12, 1998 Brow et al...... 435/6 Cuthbert, "Hepatitis C: Progress and Problems.” Clin. Microbiol. 5,874,283 A 2/1999 Harrington et al...... 435/252 Rev. 7:505-532 (1994). 5,882,867 A 3, 1999 Ullman et al...... 435/6 DeMott et al., “Human RAD2 Homolog 1 5'-3'-Exo/Endonuclease

5,888,780 A 3/1999 Dahlberg et al. . 435/91.53 Can Efficiently Excise a Displaced DNA Fragment Containing a 5,981,176 A 11/1999 Wallace ...... 435/6 5'-Terminal Abasic Lesion by Endonuclease Activity,” J. Biol. 5,985,557 A 11/1999 Prudent et al. ... 435/6 Chem. 271:30068-30076 (1996). 5,994,069 A * 11/1999 Hall et al...... 435/6 Doty et al., “Strand Separation and Specific Recombination in 6,001,567 A 12/1999 Brow et al...... 435/6 Deoxyribonucleic Acids: Physical Chemical Studies.” Proc. Natl. 6,458,535 B1 * 10/2002 Hall et al...... 435/6 Acad. Sci. USA 46:461-476 (1960). 6,913,881 B1* 7/2005 Aizenstein et al...... 435/6 Duck et al., “Probe Amplifier System Based on Chimeric Cycling 7, 195,871 B2 * 3/2007 Lyamichev et al...... 435/6 Oligonucleotides,” BioTech., 9: 142-147 (1990). Dunn et al., "Complete Nucleotide Sequence of Bacteriophage T7 FOREIGN PATENT DOCUMENTS DNA and the Locations of T7 Genetic Elements.' J. Mol. Biol. EP O 482 714 A1 10, 1991 166:477-535 (1983). 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Erlich, Ed.), Stockton Press, New York, p. 19 Nucleic Acid Hybridization, Eds Hames & Higgins, IRL Press, (1989). Washington, DC, pp. 73-111 (1985). Guatelli et al., “Isothermal, in vitro amplification of nucleic acids by Electrophoresis, 2nd Edition, ed. Anthony T. Andrews, Clarendon a multienzyme reaction modeled after retroviral replication.” Proc. Press, New York, New York (1986), pp. 153-154. Natl. Acad. Sci., 87: 1874-1878 (1990) with an erratum at Proc. Natl. Antao et al. "Athermodynamic study of unusually stable RNA and Acad. Sci., 87: 7797 (1990). DNA hairpins.” Nucl. Acids Res. 19:5901-5905 (1991). Harrington et al., “DNA Structural Elements Required for FEN-1 Bambara et al., “Enzymes and Reactions at the Eukaryotic DNA Binding.” J. Biol. Chem. 270:4503-4508 (1995). Replication Fork,” J. Biol. Chem. 272:4647-4650 (1997). 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(Ed.)), PCR Technology. Principles and Endonuclease RAD2 Homologue 1 or Flap Endonuclease 1", J. Applications for DNA Amplification, Stockton Press, pp. 61-70 Biol. Chem. 271:29624-29632 (1996). (1991). Bergseid et al., “A High Fidelity Thermostable DNA Polymerase Hiraro et al. “Most compact hairpin-turn structure exerted by a short Isolated from Pyrococcus furiosus, "Strategies 4:34-35 (1991). DNA fragment, d(GCGAAGC) in solution: an extraordinarily stable Bhagwat et al., “The 5'-Exonuclease Activity of Bacteriophage T4 structure resistant to nucleases and heat,” Nuc. Acids Res. 22:576 RNase H is Stimulated by the T4 Gene 32 Single-stranded DNA 582 (1994). binding Protein, but Its Flap Endonuclease Is Inhibited,” J. Biol. Holland et al., “Detection of specific polymerase chain reaction Chem. 272:28523-28530 (1997). product by utilizing the 5'-3' exonuclease activity of Thermus Bonch-Osmolovskaya, et al., Microbiology (Engl. Transl. of aquaticus DNA polymerase.” Proc. Natl. Acad. Sci. 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U.S. Patent Apr. 8, 2008 Sheet 13 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 14 of 128 US 7,354,708 B2

Cleavage Sites Y b Substrate Strand

2 W &YCGGAATTCGAGCTCGCC C S.Š KGCCTTAAGCTCGAGCG G AryKSK Template Strand SksCY \s

FIG. 5 U.S. Patent Apr. 8, 2008 Sheet 15 of 128 US 7,354,708 B2

DNAP T S TARGET

DNA M -- - M

FIG. 6 U.S. Patent Apr. 8, 2008 Sheet 16 of 128 US 7,354,708 B2

DNAP CYCLE NUMBER U.S. Patent Apr. 8, 2008 Sheet 17 Of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 18 of 128 US 7,354,708 B2

s U.S. Patent Apr. 8 , 2008 Sheet 19 Of 128 US 7 9 354,708 B2

£10|| U.S. Patent Apr. 8, 2008 Sheet 20 of 128 US 7,354,708 B2

27 nt 5' - Substrate DNA (206 nt -14G6 CAGCTATGACCATGATTACGCCAAGCT->( ) SGTCGATACTGGTACTAATG SCSM. C. 19 - 12 5' 1QQ

85 nt Substrate DNA (206 nt) 1. ^46 GCATGCCTGCAGGTCGACTCTAGAGGATCCC ->- So CGTACGGACGTCCAGCTGAGATCTCCTAGG ( QYSAvC 5 Sos 30 - 12 1QQ FIG. 1 1A

15 nt Substrate RNA (46 nt) - 21 AAGCUUGCAUGCCUGCAGGUCGACUCUAGAGGAUCCCC 3' 3' CGTACGGACGTCCAGCTGAGATCTCCTAGG 5' 30- 0

F.G. 12A U.S. Patent Apr. 8, 2008 Sheet 21 of 128 US 7,354,708 B2

FIG. 11B

FIG. 12B

U.S. Patent US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 25 Of 128 US 7,354,708 B2

Cleavage & & a. Sites 9% Y b 2 %Y(CGGAATTCGAGCTCGCC C ya SS KGCCTTAAGCTCGAGCGG KSCSQXY. es WNKScks

FIG. 15E U.S. Patent Apr. 8, 2008 Sheet 26 of 128 US 7,354,708 B2

c ? l C CMO H O C/D C/O g C/D D CCU D O U 5 - CC Q- S D 2 - 2 2 D CD O CL U -

N. -- --

r -- --

cy -- S.

sa s Her U.S. Patent Apr. 8, 2008 Sheet 27 Of 128 US 7,354,708 B2

4.l. *Galv?o?ýš? B|WÄZNE d|109dzº U.S. Patent Apr. 8, 2008 Sheet 28 of 128 US 7,354,708 B2

SITES OF CLEAVAGE (32p) A WITH A GAP OF 6 nt. 6.4c.Oc 60% 164 40% GATTTAGGGACACTATAG5' 36.46 CCGGAAT C CTTAAGCTAAATC CACTGTGATATCTTATGTGCCTTA, G 3 f

FIG. 18A

U.S. Patent Apr. 8, 2008 Sheet 31 of 128 US 7,354,708 B2

1 5' N TARGET NUCLEIC ACID (206 nt) 3'

35 ft 2 5' C leavage site TARGET NUCLEIC ACID (206 nt) 3' Jrt ITT I I I I I I I I I I I, XSYPILOT OLIGONUCLEOTIDE5

85 ?t 3. 5 Cleavage site TARGET NUCLEIC ACID (206 nt) 3' 5 ------5- PILOT OLIGONUCLEOTIDE

FIG. 20A U.S. Patent Apr. 8, 2008 Sheet 32 of 128 US 7,354,708 B2

se CN cC

U.S. Patent Apr. 8, 2008 Sheet 33 of 128 US 7,354,708 B2

1 2 3 4 5 6 7 8 a 7 b

age - 27 206 - war as - 24 85

27 24

L ENZYME:- DNAPTaq ATaq

FIG. 21 A FIG. 21B U.S. Patent Apr. 8, 2008 Sheet 34 of 128 US 7,354,708 B2

O O CN

A st SS

Air s

C - N ar CNN c5 O

U.S. Patent Apr. 8, 2008 Sheet 36 of 128 US 7,354,708 B2

N u-1

5' 3' k->

PLASMID TARGET

k =P 5'TERMINAL PHOSPHATE

FIG. 24A U.S. Patent Apr. 8, 2008 Sheet 37 Of 128 US 7,354,708 B2

FIG. 24B U.S. Patent Apr. 8, 2008 Sheet 38 of 128 US 7,354,708 B2

CoA

O - n - I -

CN X O o

LL gce D 2

2.L s O 2

U.S. Patent Apr. 8, 2008 Sheet 40 of 128 US 7,354,708 B2

-C22

-C15

FIG. 27

U.S. Patent Apr. 8, 2008 Sheet 42 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 43 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 44 of 128 US 7,354,708 B2

FIG. 31 U.S. Patent Apr . 8, 2008 Sheet 45 of 128 US 7 9 354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 46 of 128 US 7,354,708 B2

CO N g U.S. Patent Apr. 8, 2008 Sheet 47 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 48 of 128 US 7,354,708 B2

55°C 60°C

FIG. 35 U.S. Patent Apr. 8, 2008 Sheet 49 of 128 US 7,354,708 B2

ELEVATEDC CLEAVAGE AGENT

v/""CLEAVAGE STE

FIG. 36A

UPSTREAMOLEGONUCLEOTOE CLEAVAGE AGENT Y-clavaGEsCLEAVAGE SITE

FIG. 36B U.S. Patent Apr. 8, 2008 Sheet 50 of 128 US 7,354,708 B2 -*-*****eee833eeegeeggae,gaeae-909ITOB908'd

,g-poqqiq283naqoqnopon?po?2-?D3D2e234423223323432222342-,8

09ITO9NILOBHIQ39\//\\/3TDBAISWANI U.S. Patent Apr. 8, 2008 Sheet 51. Of 128 US 7,354,708 B2

FIG. 38 U.S. Patent Apr. 8, 2008 Sheet 52 of 128 US 7,354,708 B2

2 3 4 5 6 7 8 9 10 1 1 2 3 14

F.G. 39 U.S. Patent Apr. 8, 2008 Sheet 53 of 128 US 7,354,708 B2

1 2 3 4 5 6 7

narry rarylhur run vs. s. s r. ss. -- - -a arrams. # verr w vir v-as-a-r ------a-- rs sur sur - war a

FIG. 40 U.S. Patent Apr. 8, 2008 Sheet 54 of 128 US 7,354,708 B2

FIG. 41 U.S. Patent Apr. 8, 2008 Sheet 55 of 128 US 7,354,708 B2

OBAVJETON[] –3808d 0BAWETO —3.808d

U.S. Patent Apr. 8, 2008 Sheet 57 Of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 58 Of 128 US 7,354,708 B2

(s,Lou?uueOLO)OZ. (s,Lou?ue9O)#7/ U.S. Patent Apr. 8, 2008 Sheet 59 of 128 US 7,354,708 B2

H0 9/ U.S. Patent Apr. 8, 2008 Sheet 60 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 61 of 128 US 7,354,708 B2

70 74 75 76 SAMPLE V7 Odp' 74 dop' 7500/bdp 760ip LOADINGPOSITION

FIG. 48

U.S. Patent Apr. 8, 2008 Sheet 63 of 128 US 7,354,708 B2

5O 55 60 65 -

U.S. Patent Apr. 8, 2008 Sheet 64 of 128 US 7,354,708 B2

F.G. 50 U.S. Patent Apr. 8, 2008 Sheet 65 of 128 US 7,354,708 B2

ºÈ?zzzzzzzzzzzz 1 4 DOEL),:ZZZZZZZZZZZZZZZZZZ? ZZZZXZZZZZZZZZ

F.G. 51 U.S. Patent Apr. 8, 2008 Sheet 66 of 128 US 7,354,708 B2

s U.S. Patent Apr. 8, 2008 Sheet 67 of 128 US 7,354,708 B2

PRIMER - + C T A G

* is - E., B., , ,

*" .. . r . ; * * , ... sai asis .. ... r. s. sr. . wri- is al creas

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ses

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FIG. 53

U.S. Patent Apr. 8, 2008 Sheet 69 of 128 US 7,354,708 B2

G Et GH 9. G HN B GD Ph 30lgG) NNH3 GE) -----5N 1- G) 3 3C N Ph N Ph H aOnh HN 3. s HN () w 4c.2 E. 2 NH2 NH2 BROMIDE (1,3-PROPANEDIAMINO)- (DIETHYLENETRAMINO)- PROPOUM PROPDIUM Mo M M IMG 2Ir-NYAMG e-I, 216e rol-9-a- N N' Mo N Me M Cs CS o Mé N &y Mé N y Mé N (N-N'-TETRAMETHYL- (N-N'-TETRAMETHYL THAZOLE 1,2-ETHANEDAMNO)- 1,3-PROPANEDIAMINO)- ORANGE PROPYL THAZOLE ORANGE PROPYL THAZOLE ORANGE o Mo Mo Mo o: S 9-8 G.S.c M.a E. M. GS-2M. s S ad N SS 4C s S

Mo Q) isN Mo R) TOTAB Mo TOTO Rege G) Relie hNSN Pha-e-NSPh H.N. Ph e N 2 el 4cGd Oa o,O 4C n & S NH NH2 NH2 N-y 2 TOED1 (RH) Eth TOED2 (R-CH, M

H e2 "DS F.G. 55 a?? CO 5. U.S. Patent Apr. 8, 2008 Sheet 70 of 128 US 7,354,708 B2

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U.S. Patent Apr. 8, 2008 Sheet 72 of 128 US 7,354,708 B2

FIG. 58

U.S. Patent Apr. 8, 2008 Sheet 78 of 128 US 7,354,708 B2

S-33 5. CLEAVAGESITE G G G cGCTGTCTCGCT A coacAGAGCG, 3

GC 11 - 8 - O CT CLEAVAGE SITE CT G GYCGCTGTCT G A GCGACAGA 3 A

F.G. 60 U.S. Patent Apr. 8, 2008 Sheet 79 of 128 US 7,354,708 B2

S3 - UNCUT

... -- CLEAVED U.S. Patent Apr. 8, 2008 Sheet 80 of 128 US 7,354,708 B2

- S-60 UNCUT

F.G. 62 U.S. Patent Apr. 8, 2008 Sheet 81 of 128 US 7,354,708 B2

1 2

sai SEsia's

3- CIRCULAR -- LINEAR

FIG. 63 U.S. Patent Apr. 8, 2008 Sheet 82 of 128 US 7,354,708 B2

i cus

KD - i 97.4 -

66.2 - 55.0 - 42.7 - 40.0 -

31.0 -

F.G. 64 U.S. Patent Apr. 8, 2008 Sheet 83 of 128 US 7,354,708 B2

op2 (6) C/D CC D c ENZYME - CD &

FIG. 65 U.S. Patent Apr. 8, 2008 Sheet 84 of 128 US 7,354,708 B2

-H a -- - -- TERMINAL TRANSFERASE - - -- ALKALINE PHOSPHATASE + + + it THERMAL DEGRADATION

5'-nAGAAAggaagggaagaaag.cgaaagG F.G. 66 U.S. Patent Apr. 8, 2008 Sheet 85 of 128 US 7,354,708 B2

arr

- - - a s aS ". re. artyar . B s a . . . as au r A. t r-r ... U.S. Patent Apr. 8, 2008 Sheet 86 of 128 US 7,354,708 B2

-serpre-ses-----sourosawaarease arrier-rrrr", "r"

- 8 o

O s to 2. O . S st s 3 O s 3 P

- isgs 2 SS O S

4. wrx vs 'star U.S. Patent Apr. 8, 2008 Sheet 87 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 88 of 128 US 7,354,708 B2

CN U.S. Patent Apr. 8, 2008 Sheet 89 of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 90 Of 128 US 7,354,708 B2

8| U.S. Patent Apr. 8, 2008 Sheet 91. Of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 92 Of 128 US 7,354,708 B2

U.S. Patent Apr. 8, 2008 Sheet 94 of 128 US 7,354,708 B2

1 BASE 1 BASE 1 BASE ALL COMP MSMATCHALL COMPM ISMATCH ALL COMP MSMATCH 2N. 2N 1NP 1NP

NVADER 67 114.l.115 116 112 113

520C V17

FIG. 75A 1 BASE BASE 1 BASE ALL COMPM ISMATCH ALL COMP MISMATCH ALL COMP MSMATCH | 2N 2N 1NP 1NP NVADER 67 114 ... 115. 116.112. 13 # 2 3 4 5 6

55°C V1806 V1221 V10

FIG. 75B 1 BASE BASE 1 BASE ALL COMP MISMATCHALL COMP MISMATCH AL COMP MSMATCH 2N 2N. 1NP 1NP NWADER 67 114 115 116 112 113 6

580C w N V2 V5. V5 V2

... t assissass-as-ad'...i.s: ... . .x.3. * is or . . . s: 3:...* ...... :

U.S. Patent Apr. 8, 2008 Sheet 96 of 128 US 7,354,708 B2

TEMP -o- 47o 50o 53o 56,o TARGET -- 165 166 165 166 165. 166 165 166

FIG. 77