Tuesday, July 29, 2008

PCR primers sequences

Human papillomavirus (HPV) capture probe and PCR primers sequences.
6 capture CAG AAT TGG TGT ATG TGG AAG A(N152)
11 capture TAA TCT GAA TTA GTG TAT GTA GCA GAT TTA GAC A(N152)
16 capture GTA GTT TCT GAA GTA GAT ATG G(N152)
18 capture TGG TAG CAT CAT ATT GCC CAG G(N152)
26 capture ATC AGA TGG TTT AAA TGG AGT GGA TGC(N152)
31 capture TAC TAC TTT TAA ATG TAG TAT CAC(N152)
35 capture ACT GTC ACT AGA AGA CAC AGC AGA ACA CA(N152)
40 capture GGG GGA CTG TGT GGC ACC A(N152)
42 capture AGC AGC TGT ATA TGT ATC ACC AGA TGT TGC AGT GGC TCA(N152)
45 capture CTT AGT AGG GTC ATA TGT ACT TGG C(N152)
51 capture TTG GGG AAA CCG CAG CAG TGG CAG GGC TA(N152)
52 capture TAT GTG CTT TCC TTT TTA ACC T(N152)
54 capture GTC AGA ATT ATT AAA GCT ATC CTG CG(N152)
56 capture TTT TCG TGC ATC ATA TTT ACT TA(N152)
58 capture GTA CCT TCC TTA GTT ACT TCA G(N152)
59 capture CTG GTA GGT GTG TAT ACA TTA G(N152)
66 capture CAC GGG CAT CAT ATT TAG TTA A(N152)
68 capture TTA AAT TTA TTA GGA TCA TAA ATA TTT GGT A(N152)
82 capture AAT GTT TGT GCA ACA GAT TGA GTA ACA GCT GTG(N152)
83 capture GTT AGA GGC TGT GTA TTC ATT AGC(N152)
84 capture GGT TTA TAT TCT GAT TCG GTG T(N152)
globin capture AGC AAT AGA TGG CTC TGC CC(N152)
globin primer GAA GAG CCA AGG ACA GGT AC
globin rev primer BIOTIN CAA CTT CAT CCA CGT TCA CC
HMB01 rev primer BIOTIN GCG ACC CAA TGC AAA TTG GT
PGMY09F primer BIOTIN CGT CCC AAA GGA AAC TGA TC
PGMY09G primer BIOTIN CGA CCT AAA GGA AAC TGA TC
PGMY09H primer BIOTIN CGT CCA AAA GGA AAC TGA TC
PGMY09I primer BIOTIN GCC AAG GGG AAA CTG ATC
PGMY09J primer BIOTIN CGT CCC AAA GGA TAC TGA TC
PGMY09K primer BIOTIN CGT CCA AGG GGA TAC TGA TC
PGMY09L primer BIOTIN CGA CCT AAA GGG AAT TGA TC
PGMY09M primer BIOTIN CGA CCT AGT GGA AAT TGA TC
PGMY09N primer BIOTIN CGA CCA AGG GGA TAT TGA TC
PGMY09P primer BIOTIN GCC CAA CGG AAA CTG ATC
PGMY09Q primer BIOTIN CGA CCC AAG GGA AAC TGG TC
PGMY09R primer BIOTIN CGT CCT AAA GGA AAC TGG TC
PGMY11A primer GCA CAG GGA CAT AAC AAT GG
PGMY11B primer GCG CAG GGC CAC AAT AAT GG
PGMY11C primer GCA CAG GGA CAT AAT AAT GG
PGMY11D primer GCC CAG GGC CAC AAC AAT GG
PGMY11F primer GCT CAG GGT TTA AAC AAT GG
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HPV - DNA COADING

Table 2: HPV type and signal probe sequence.
6 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT AGT TAC GGA TGC AC
11 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG ATG CAG ATA GTG TCA T
16 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CGC ATA ATG ACA TAT TT
18 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TAC AGG AGA CTG TGT AG
26 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) AGA TGC TGT AGA TAA TGT AC
31 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT TTG CAA TTG CAG CA
35 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG ACA TAT TTG TTC TAC GG
40 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TAA GGT TAA ATT AGT GCA ACG A
42 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAA AGA CAT GTT AGT ACT AC
45 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ACA GGA TTT TGT GTA GAG G
51 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ATA GTT AAA TTT GTA CTT CTG G
52 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CAC ATA AAG TCA TG
54 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGG ATG CTG TAG CAC AC
56 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) ACT GTT CTG TAG CAG TAC T
58 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGC ATA ATG TCA TAT TAG TG
59 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) GAA TAG AAG AAG TAG TAG AA
66 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGT GCT TTT AGC TGC AT
68 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CAG CTG ATT CAG TAG TAG TA
82 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CTA ATG GTT AAA TTG GTA GTT
83 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) CTG TGT AGC AGG AGC TGA AA
84 (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGG TAG CAG CAC TAA TA
Globin (N6) C(N6) G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) TGA CTT TTA TGC CCA GAC CTG G
Table 3: HPV type and target mimic oligonucleotide sequences
6 ACC ACA CGC AGT ACC AAC ATG ACA TTA TGT GCA TCC GTA ACT ACA TCT TCC ACA TAC ACC AAT TCT GAT TAT AAA
11 GTA CAA ATA TGA CAC TAT GTG CAT CTG TGT CTA AAT CTG CTA CAT ACA CTA ATT CAG ATT ATA AGG AAT ACA TGC G
16 ACT ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA ACT ACA TAT AAA AAT ACT AAC TTT AA
18 ACC ACT CCC AGT ACC AAT TTA ACA ATA TGT GCT TCT ACA CAG TCT CCT GTA CCT GGG CAA TAT GAT GCT ACC AAA T
26 GTA CTA ACC TTA CCA TTA GTA CAT TAT CTG CAG CAT CTG CAT CCA CTC CAT TTA AAC CAT CTG ATT ATA AAC AAT T
31 ACC ACA CGT AGT ACC AAT ATG TCT GTT TGT GCT GCA ATT GCA AAC AGT GAT ACT ACA TTT AAA AGT AGT AAT TTT AA
35 TGT AGT TGA TAC AAC CCG TAG TAC AAA TAT GTC TGT GTG TTC TGC TGT GTC TTC TAG TGA CAG TAC ATA TAA AAA T
40 AGT TGT AGA CAC CAC TCG TAG CAC TAA TTT AAC CTT ATG TGC TGC CAC ACA GTC CCC CAC ACC AAC CCC ATA TAA T
42 ACT ACC CGT AGT ACT AAC ATG ACT TTG TGT GCC ACT GCA ACA TCT GGT GAT ACA TAT ACA GCT GCT AAT TTT AAG G
45 AAC ATT ATG TGC CTC TAC ACA AAA TCC TGT GCC AAG TAC ATA TGA CCC TAC TAA GTT TAA GCA GTA TAG TAG ACA T
51 GTT GAT ACT ACC AGA AGT ACA AAT TTA ACT ATT AGC ACT GCC ACT GCT GCG GTT TCC CCA ACA TTT ACT CCA AGT A
52 ACC ACT CGT AGC ACT AAC ATG ACT TTA TGT GCT GAG GTT AAA AAG GAA AGC ACA TAT AAA AAT GAA AAT TTT AAG
54 GTA CTA ACC TAA CAT TGT GTG CTA CAG CAT CCA CGC AGG ATA GCT TTA ATA ATT CTG ACT TTA GGG AGT ATA TTA G
56 GTA CTA ACA TGA CTA TTA GTA CTG CTA CAG AAC AGT TAA GTA AAT ATG ATG CAC GAA AAA TTA ATC AGT ACC TTA G
58 ACC ACT CGT AGC ACT AAT ATG ACA TTA TGC ACT GAA GTA ACT AAG GAA GGT ACA TAT AAA AAT GAT AAT TTT AAG
59 ACT ACT CGC AGC ACC AAT CTT TCT GTG TGT GCT TCT ACT ACT TCT TCT ATT CCT AAT GTA TAC ACA CCT ACC AGT
66 ACT ACC AGA AGC ACC AAC ATG ACT ATT AAT GCA GCT AAA AGC ACA TTA ACT AAA TAT GAT GCC CGT GAA ATC AAT
68 GTA CCA ATT TTA CTT TGT CTA CTA CTA CTG AAT CAG CTG TAC CAA ATA TTT ATG ATC CTA ATA AAT TTA AGG AAT A
82 ACT ACT AGA AGT ACC AAT TTA ACC ATT AGC ACT GCT GTT ACT CAA TCT GTT GCA CAA ACA TTT ACT CCA GCA AAC T
83 GTA CCA ATA TTA CTA TTT CAG CTG CTG CTA CAC AGG CTA ATG AAT ACA CAG CCT CTA ACT TTA AGG AAT ACC TCC G
84 ACC ACC CGC AGC ACC AAT TTT ACT ATT AGT GCT GCT ACC AAC ACC GAA TCA GAA TAT AAA CCT ACC AAT TTT AAG
globin GGG AGG GCA GGA GCC AGG GCT GGG CAT AAA AGT CAG GGC AGA GCC ATC TAT TGC TTA CAT TTG CTT CTG ACA CAA C
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HPV Leavel

Many of the HPV target mimics tested resulted in a specific
signal, with the exception of HPV types 16, 26, 35, 40, 42,
51, 52, and 82. This nonspecific hybridization signal generally
resulted from a long stretch of complementary
sequences (6–8 bases) among the HPV types due to their
close genetic relatedness. Nonspecific hybridization was
eliminated by introducing a mismatched base to the complementary
region in the signal probes for HPV types 16,
26, 35, 42, and 82 and the capture probes for HPV types
40, 42, 51, 52, and 82. For instance, the HPV 16 signal
probe hybridized with both the HPV 16 target mimic (Fig.
3, left panel) and the HPV 52 capture probe (Fig. 3, center
panel). This cross-reactivity was eliminated by changing
the second A base in the 5'-end of signal probe to a G base
(Fig. 3, right panel). In addition, we demonstrated that
one base modification of the signal probe (Fig. 3, left and
right panels) generated minimal negative impact to the
assay signal level.
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BIO-ELECTRONIC

Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences

specific for HPV types. Electrically conductive signal probes were synthesized to be

complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1

region that was amplified by using consensus primers served as target DNA. The amplified

target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA

chip to allow for hybridization with complementary capture probes.
Results

Two bioelectric chips were designed and successfully detected 86% of the HPV types contained

in clinical samples.
Conclusions

This model system demonstrates the potential of the eSensor platform for rapid and

integrated detection of multiple pathogens.
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BioElectronic Will Chance Our Lifestyle......

Global emergence of pathogenic infectious diseases by both natural and intentional means

presents a formidable challenge to infectious disease surveillance and response, namely

timely and efficient pathogen detection. Many laboratory methods exist for identifying

pathogens, but most require exquisite care in sample handling and processing prior to

characterization of a pathogen. In addition, costly and perishable reagents, equipment, and

supplies are required for sensitive and specific detection. The ideal detection system would

integrate sample processing and pathogen characterization into a single automated device

that would eliminate laborious, and time consuming sample processing and costly detection.

Bioelectronic detection of nucleic acids on a miniature solid support is one of the first

steps toward development of such an integrated detection device.
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Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.