DNA sequencing methods

1
DNA sequencing methods
2
Introduction
Developing new methods and instruments that
permit fast polynucleotide sequencing has
attracted considerable attention recently. A
device for rapid DNA decryption will allow quick
identification of pathogens to save many lives
during an epidemic or a bioterrorist attack.
Moreover, doctors would be able to diagnose a
disease, judge possible risks, and design a
special treatment plan based on knowledge about
which disease-related genes a patient carries.
Although DNA sequencing has important medical
applications, present methods in sequencing
polynucleotides are slow, costly, and inaccurate.
The prior sequencing method proposed by Fredrick
Sanger is performed by replicating DNA under
control to obtain fragments with various lengths
such that the complete DNA sequence can be
derived from these fragments using gel
electrophoresis. However long fragments (gt 500
nucleotides) cannot be sequenced at one go since
the mobility of long fragments are independent of
their length. An analogy of using the Sanger
method to sequence human DNA is to cut a 1 meter
long noodle into 10 million fragments and then
put them back in the right order, which is a
difficult task. Thus the human genome project
cost 3 billion US dollars and took 13 years to
complete the draft of human DNA.
3
SARS code
CTACCCAGGAAAAGCCAACCAACCTCGATCTCTTGTAGATCTGTTCTCTA
AACGAACTTTAAAATCTGTGTAGCTGTCGCTCGGCTGCATGCCTAGTGCA
CCTACGCAGTATAAACAATAATAAATTTTACTGTCGTTGACAAGAAACGA
GTAACTCGTCCCTCTTCTGCAGACTGCTTACGGTTTCGTCCGTGTTGCAG
TCGATCATCAGCATACCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGA
TGGAGAGCCTTGTTCTTGGTGTCAACGAGAAAACACACGTCCAACTCAGT
TTGCCTGTCCTTCAGGTTAGAGACGTGCTAGTGCGTGGCTTCGGGGACTC
TGTGGAAGAGGCCCTATCGGAGGCACGTGAACACCTCAAAAATGGCACTT
GTGGTCTAGTAGAGCTGGAAAAAGGCGTACTGCCCCAGCTTGAACAGCCC
TATGTGTTCATTAAACGTTCTGATGCCTTAAGCACCAATCACGGCCACAA
GGTCGTTGAGCTGGTTGCAGAAATGGACGGCATTCAGTACGGTCGTAGCG
GTATAACACTGGGAGTACTCGTGCCACATGTGGGCGAAACCCCAATTGCA
TACCGCAATGTTCTTCTTCGTAAGAACGGTAATAAGGGAGCCGGTGGTCA
TAGCTATGGCATCGATCTAAAGTCTTATGACTTAGGTGACGAGCTTGGCA
CTGATCCCATTGAAGATTATGAACAAAACTGGAACACTAAGCATGGCAGT
GGTGCACTCCGTGAACTCACTCGTGAGCTCAATGGAGGTGCAGTCACTCG
CTATGTCGACAACAATTTCTGTGGCCCAGATGGGTACCCTCTTGATTGCA
TCAAAGATTTTCTCGCACGCGCGGGCAAGTCAATGTGCACTCTTTCCGAA
CAACTTGATTACATCGAGTCGAAGAGAGGTGTCTACTGCTGCCGTGACCA
TGAGCATGAAATTGCCTGGTTCACTGAGCGCTCTGATAAGAGCTACGAGC
ACCAGACACCCTTCGAAATTAAGAGTGCCAAGAAATTTGACACTTTCAAA
GGGGAATGCCCAAAGTTTGTGTTTCCTCTTAACTCAAAAGTCAAAGTCAT
TCAACCACGTGTTGAAAAGAAAAAGACTGAGGGTTTCATGGGGCGTATAC
GCTCTGTGTACCCTGTTGCATCTCCACAGGAGTGTAACAATATGCACTTG
TCTACCTTGATGAAATGTAATCATTGCGATGAAGTTTCATGGCAGACGTG
CGACTTTCTGAAAGCCACTTGTGAACATTGTGGCACTGAAAATTTAGTTA
TTGAAGGACCTACTACATGTGGGTACCTACCTACTAATGCTGTAGTGAAA
ATGCCATGTCCTGCCTGTCAAGACCCAGAGATTGGACCTGAGCATAGTGT
TGCAGATTATCACAACCACTCAAACATTGAAACTCGACTCCGCAAGGGAG
GTAGGACTAGATGTTTTGGAGGCTGTGTGTTTGCCTATGTTGGCTGCTAT
AATAAGCGTGCCTACTGGGTTCCTCGTGCTAGTGCTGATATTGGCTCAGG
CCATACTGGCATTACTGGTGACAATGTGGAGACCTTGAATGAGGATCTCC
TTGAGATACTGAGTCGTGAACGTGTTAACATTAACATTGTTGGCGATTTT
CATTTGAATGAAGAGGTTGCCATCATTTTGGCATCTTTCTCTGCTTCTAC
AAGTGCCTTTATTGACACTATAAAGAGTCTTGATTACAAGTCTTTCAAAA
CCATTGTTGAGTCCTGCGGTAACTATAAAGTTACCAAGGGAAAGCCCGTA
AAAGGTGCTTGGAACATTGGACAACAGAGATCAGTTTTAACACCACTGTG
TGGTTTTCCCTCACAGGCTGCTGGTGTTATCAGATCAATTTTTGCGCGCA
CACTTGATGCAGCAAACCACTCAATTCCTGATTTGCAAAGAGCAGCTGTC
ACCATACTTGATGGTATTTCTGAACAGTCATTACGTCTTGTCGACGCCAT
GGTTTATACTTCAGACCTGCTCACCAACAGTGTCATTATTATGGCATATG
TAACTGGTGGTCTTGTACAACAGACTTCTCAGTGGTTGTCTAATCTTTTG
GGCACTACTGTTGAAAAACTCAGGCCTATCTTTGAATGGATTGAGGCGAA
ACTTAGTGCAGGAGTTGAATTTCTCAAGGATGCTTGGGAGATTCTCAAAT
TTCTCATTACAGGTGTTTTTGACATCGTCAAGGGTCAAATACAGGTTGCT
TCAGATAACATCAAGGATTGTGTAAAATGCTTCATTGATGTTGTTAACAA
GGCACTCGAAATGTGCATTGATCAAGTCACTATCGCTGGCGCAAAGTTGC
GATCACTCAACTTAGGTGAAGTCTTCATCGCTCAAAGCAAGGGACTTTAC
CGTCAGTGTATACGTGGCAAGGAGCAGCTGCAACTACTCATGCCTCTTAA
GGCACCAAAAGAAGTAACCTTTCTTGAAGGTGATTCACATGACACAGTAC
TTACCTCTGAGGAGGTTGTTCTCAAGAACGGTGAACTCGAAGCACTCGAG
ACGCCCGTTGATAGCTTCACAAATGGAGCTATCGTCGGCACACCAGTCTG
TGTAAATGGCCTCATGCTCTTAGAGATTAAGGACAAAGAACAATACTGCG
CATTGTCTCCTGGTTTACTGGCTACAAACAATGTCTTTCGCTTAAAAGGG
GGTGCACCAATTAAAGGTGTAACCTTTGGAGAAGATACTGTTTGGGAAGT
TCAAGGTTACAAGAATGTGAGAATCACATTTGAGCTTGATGAACGTGTTG
ACAAAGTGCTTAATGAAAAGTGCTCTGTCTACACTGTTGAATCCGGTACC
GAAGTTACTGAGTTTGCATGTGTTGTAGCAGAGGCTGTTGTGAAGACTTT
ACAACCAGTTTCTGATCTCCTTACCAACATGGGTATTGATCTTGATGAGT
GGAGTGTAGCTACATTCTACTTATTTGATGATGCTGGTGAAGAAAACTTT
TCATCACGTATGTATTGTTCCTTTTACCCTCCAGATGAGGAAGAAGAGGA
CGATGCAGAGTGTGAGGAAGAAGAAATTGATGAAACCTGTGAACATGAGT
ACGGTACAGAGGATGATTATCAAGGTCTCCCTCTGGAATTTGGTGCCTCA
GCTGAAACAGTTCGAGTTGAGGAAGAAGAAGAGGAAGACTGGCTGGATGA
TACTACTGAGCAATCAGAGATTGAGCCAGAACCAGAACCTACACCTGAAG
AACCAGTTAATCAGTTTACTGGTTATTTAAAACTTACTGACAATGTTGCC
ATTAAATGTGTTGACATCGTTAAGGAGGCACAAAGTGCTAATCCTATGGT
GATTGTAAATGCTGCTAACATACACCTGAAACATGGTGGTGGTGTAGCAG
GTGCACTCAACAAGGCAACCAATGGTGCCATGCAAAAGGAGAGTGATGAT
TACATTAAGCTAAATGGCCCTCTTACAGTAGGAGGGTCTTGTTTGCTTTC
TGGACATAATCTTGCTAAGAAGTGTCTGCATGTTGTTGGACCTAACCTAA
ATGCAGGTGAGGACATCCAGCTTCTTAAGGCAGCATATGAAAATTTCAAT
TCACAGGACATCTTACTTGCACCATTGTTGTCAGCAGGCATATTTGGTGC
TAAACCACTTCAGTCTTTACAAGTGTGCGTGCAGACGGTTCGTACACAGG
TTTATATTGCAGTCAATGACAAAGCTCTTTATGAGCAGGTTGTCATGGAT
TATCTTGATAACCTGAAGCCTAGAGTGGAAGCACCTAAACAAGAGGAGCC
ACCAAACACAGAAGATTCCAAAACTGAGGAGAAATCTGTCGTACAGAAGC
CTGTCGATGTGAAGCCAAAAATTAAGGCCTGCATTGATGAGGTTACCACA
ACACTGGAAGAAACTAAGTTTCTTACCAATAAGTTACTCTTGTTTGCTGA
TATCAATGGTAAGCTTTACCATGATTCTCAGAACATGCTTAGAGGTGAAG
ATATGTCTTTCCTTGAGAAGGATGCACCTTACATGGTAGGTGATGTTATC
ACTAGTGGTGATATCACTTGTGTTGTAATACCCTCCAAAAAGGCTGGTGG
CACTACTGAGATGCTCTCAAGAGCTTTGAAGAAAGTGCCAGTTGATGAGT
ATATAACCACGTACCCTGGACAAGGATGTGCTGGTTATACACTTGAGGAA
GCTAAGACTGCTCTTAAGAAATGCAAATCTGCATTTTATGTACTACCTTC
AGAAGCACCTAATGCTAAGGAAGAGATTCTAGGAACTGTATCCTGGAATT
TGAGAGAAATGCTTGCTCATGCTGAAGAGACAAGAAAATTAATGCCTATA
TGCATGGATGTTAGAGCCATAATGGCAACCATCCAACGTAAGTATAAAGG
AATTAAAATTCAAGAGGGCATCGTTGACTATGGTGTCCGATTCTTCTTTT
ATACTAGTAAAGAGCCTGTAGCTTCTATTATTACGAAGCTGAACTCTCTA
AATGAGCCGCTTGTCACAATGCCAATTGGTTATGTGACACATGGTTTTAA
TCTTGAAGAGGCTGCGCGCTGTATGCGTTCTCTTAAAGCTCCTGCCGTAG
TGTCAGTATCATCACCAGATGCTGTTACTACATATAATGGATACCTCACT
TCGTCATCAAAGACATCTGAGGAGCACTTTGTAGAAACAGTTTCTTTGGC
TGGCTCTTACAGAGATTGGTCCTATTCAGGACAGCGTACAGAGTTAGGTG
TTGAATTTCTTAAGCGTGGTGACAAAATTGTGTACCACACTCTGGAGAGC
CCCGTCGAGTTTCATCTTGACGGTGAGGTTCTTTCACTTGACAAACTAAA
GAGTCTCTTATCCCTGCGGGAGGTTAAGACTATAAAAGTGTTCACAACTG
TGGACAACACTAATCTCCACACACAGCTTGTGGATATGTCTATGACATAT
GGACAGCAGTTTGGTCCAACATACTTGGATGGTGCTGATGTTACAAAAAT
TAAACCTCATGTAAATCATGAGGGTAAGACTTTCTTTGTACTACCTAGTG
ATGACACACTACGTAGTGAAGCTTTCGAGTACTACCATACTCTTGATGAG
AGTTTTCTTGGTAGGTACATGTCTGCTTTAAACCACACAAAGAAATGGAA
ATTTCCTCAAGTTGGTGGTTTAACTTCAATTAAATGGGCTGATAACAATT
GTTATTTGTCTAGTGTTTTATTAGCACTTCAACAGCTTGAAGTCAAATTC
AATGCACCAGCACTTCAAGAGGCTTATTATAGAGCCCGTGCTGGTGATGC
TGCTAACTTTTGTGCACTCATACTCGCTTACAGTAATAAAACTGTTGGCG
AGCTTGGTGATGTCAGAGAAACTATGACCCATCTTCTACAGCATGCTAAT
TTGGAATCTGCAAAGCGAGTTCTTAATGTGGTGTGTAAACATTGTGGTCA
GAAAACTACTACCTTAACGGGTGTAGAAGCTGTGATGTATATGGGTACTC
TATCTTATGATAATCTTAAGACAGGTGTTTCCATTCCATGTGTGTGTGGT
CGTGATGCTACACAATATCTAGTACAACAAGAGTCTTCTTTTGTTATGAT
GTCTGCACCACCTGCTGAGTATAAATTACAGCAAGGTACATTCTTATGTG
CGAATGAGTACACTGGTAACTATCAGTGTGGTCATTACACTCATATAACT
GCTAAGGAGACCCTCTATCGTATTGACGGAGCTCACCTTACAAAGATGTC
AGAGTACAAAGGACCAGTGACTGATGTTTTCTACAAGGAAACATCTTACA
CTACAACCATCAAGCCTGTGTCGTATAAACTCGATGGAGTTACTTACACA
GAGATTGAACCAAAATTGGATGGGTATTATAAAAAGGATAATGCTTACTA
TACAGAGCAGCCTATAGACCTTGTACCAACTCAACCATTACCAAATGCGA
GTTTTGATAATTTCAAACTCACATGTTCTAACACAAAATTTGCTGATGAT
TTAAATCAAATGACAGGCTTCACAAAGCCAGCTTCACGAGAGCTATCTGT
CACATTCTTCCCAGACTTGAATGGCGATGTAGTGGCTATTGACTATAGAC
ACTATTCAGCGAGTTTCAAGAAAGGTGCTAAATTACTGCATAAGCCAATT
GTTTGGCACATTAACCAGGCTACAACCAAGACAACGTTCAAACCAAACAC
TTGGTGTTTACGTTGTCTTTGGAGTACAAAGCCAGTAGATACTTCAAATT
CATTTGAAGTTCTGGCAGTAGAAGACACACAAGGAATGGACAATCTTGCT
TGTGAAAGTCAACAACCCACCTCTGAAGAAGTAGTGGAAAATCCTACCAT
ACAGAAGGAAGTCATAGAGTGTGACGTGAAAACTACCGAAGTTGTAGGCA
ATGTCATACTTAAACCATCAGATGAAGGTGTTAAAGTAACACAAGAGTTA
GGTCATGAGGATCTTATGGCTGCTTATGTGGAAAACACAAGCATTACCAT
TAAGAAACCTAATGAGCTTTCACTAGCCTTAGGTTTAAAAACAATTGCCA
CTCATGGTATTGCTGCAATTAATAGTGTTCCTTGGAGTAAAATTTTGGCT
TATGTCAAACCATTCTTAGGACAAGCAGCAATTACAACATCAAATTGCGC
TAAGAGATTAGCACAACGTGTGTTTAACAATTATATGCCTTATGTGTTTA
CATTATTGTTCCAATTGTGTACTTTTACTAAAAGTACCAATTCTAGAATT
AGAGCTTCACTACCTACAACTATTGCTAAAAATAGTGTTAAGAGTGTTGC
TAAATTATGTTTGGATGCCGGCATTAATTATGTGAAGTCACCCAAATTTT
CTAAATTGTTCACAATCGCTATGTGGCTATTGTTGTTAAGTATTTGCTTA
GGTTCTCTAATCTGTGTAACTGCTGCTTTTGGTGTACTCTTATCTAATTT
TGGTGCTCCTTCTTATTGTAATGGCGTTAGAGAATTGTATCTTAATTCGT
CTAACGTTACTACTATGGATTTCTGTGAAGGTTCTTTTCCTTGCAGCATT
TGTTTAAGTGGATTAGACTCCCTTGATTCTTATCCAGCTCTTGAAACCAT
TCAGGTGACGATTTCATCGTACAAGCTAGACTTGACAATTTTAGGTCTGG
CCGCTGAGTGGGTTTTGGCATATATGTTGTTCACAAAATTCTTTTATTTA
TTAGGTCTTTCAGCTATAATGCAGGTGTTCTTTGGCTATTTTGCTAGTCA
TTTCATCAGCAATTCTTGGCTCATGTGGTTTATCATTAGTATTGTACAAA
TGGCACCCGTTTCTGCAATGGTTAGGATGTACATCTTCTTTGCTTCTTTC
TACTACATATGGAAGAGCTATGTTCATATCATGGATGGTTGCACCTCTTC
GACTTGCATGATGTGCTATAAGCGCAATCGTGCCACACGCGTTGAGTGTA
CAACTATTGTTAATGGCATGAAGAGATCTTTCTATGTCTATGCAAATGGA
GGCCGTGGCTTCTGCAAGACTCACAATTGGAATTGTCTCAATTGTGACAC
ATTTTGCACTGGTAGTACATTCATTAGTGATGAAGTTGCTCGTGATTTGT
CACTCCAGTTTAAAAGACCAATCAACCCTACTGACCAGTCATCGTATATT
GTTGATAGTGTTGCTGTGAAAAATGGCGCGCTTCACCTCTACTTTGACAA
GGCTGGTCAAAAGACCTATGAGAGACATCCGCTCTCCCATTTTGTCAATT
TAGACAATTTGAGAGCTAACAACACTAAAGGTTCACTGCCTATTAATGTC
ATAGTTTTTGATGGCAAGTCCAAATGCGACGAGTCTGCTTCTAAGTCTGC
TTCTGTGTACTACAGTCAGCTGATGTGCCAACCTATTCTGTTGCTTGACC
AAGCTCTTGTATCAGACGTTGGAGATAGTACTGAAGTTTCCGTTAAGATG
TTTGATGCTTATGTCGACACCTTTTCAGCAACTTTTAGTGTTCCTATGGA
AAAACTTAAGGCACTTGTTGCTACAGCTCACAGCGAGTTAGCAAAGGGTG
TAGCTTTAGATGGTGTCCTTTCTACATTCGTGTCAGCTGCCCGACAAGGT
GTTGTTGATACCGATGTTGACACAAAGGATGTTATTGAATGTCTCAAACT
TTCACATCACTCTGACTTAGAAGTGACAGGTGACAGTTGTAACAATTTCA
TGCTCACCTATAATAAGGTTGAAAACATGACGCCCAGAGATCTTGGCGCA
TGTATTGACTGTAATGCAAGGCATATCAATGCCCAAGTAGCAAAAAGTCA
CAATGTTTCACTCATCTGGAATGTAAAAGACTACATGTCTTTATCTGAAC
AGCTGCGTAAACAAATTCGTAGTGCTGCCAAGAAGAACAACATACCTTTT
AGACTAACTTGTGCTACAACTAGACAGGTTGTCAATGTCATAACTACTAA
AATCTCACTCAAGGGTGGTAAGATTGTTAGTACTTGTTTTAAACTTATGC
TTAAGGCCACATTATTGTGCGTTCTTGCTGCATTGGTTTGTTATATCGTT
ATGCCAGTACATACATTGTCAATCCATGATGGTTACACAAATGAAATCAT
TGGTTACAAAGCCATTCAGGATGGTGTCACTCGTGACATCATTTCTACTG
ATGATTGTTTTGCAAATAAACATGCTGGTTTTGACGCATGGTTTAGCCAG
CGTGGTGGTTCATACAAAAATGACAAAAGCTGCCCTGTAGTAGCTGCTAT
CATTACAAGAGAGATTGGTTTCATAGTGCCTGGCTTACCGGGTACTGTGC
TGAGAGCAATCAATGGTGACTTCTTGCATTTTCTACCTCGTGTTTTTAGT
GCTGTTGGCAACATTTGCTACACACCTTCCAAACTCATTGAGTATAGTGA
TTTTGCTACCTCTGCTTGCGTTCTTGCTGCTGAGTGTACAATTTTTAAGG
ATGCTATGGGCAAACCTGTGCCATATTGTTATGACACTAATTTGCTAGAG
GGTTCTATTTCTTATAGTGAGCTTCGTCCAGACACTCGTTATGTGCTTAT
GGATGGTTCCATCATACAGTTTCCTAACACTTACCTGGAGGGTTCTGTTA
GAGTAGTAACAACTTTTGATGCTGAGTACTGTAGACATGGTACATGCGAA
AGGTCAGAAGTAGGTATTTGCCTATCTACCAGTGGTAGATGGGTTCTTAA
TAATGAGCATTACAGAGCTCTATCAGGAGTTTTCTGTGGTGTTGATGCGA
TGAATCTCATAGCTAACATCTTTACTCCTCTTGTGCAACCTGTGGGTGCT
TTAGATGTGTCTGCTTCAGTAGTGGCTGGTGGTATTATTGCCATATTGGT
GACTTGTGCTGCCTACTACTTTATGAAATTCAGACGTGTTTTTGGTGAGT
ACAACCATGTTGTTGCTGCTAATGCACTTTTGTTTTTGATGTCTTTCACT
ATACTCTGTCTGGTACCAGCTTACAGCTTTCTGCCGGGAGTCTACTCAGT
CTTTTACTTGTACTTGACATTCTATTTCACCAATGATGTTTCATTCTTGG
CTCACCTTCAATGGTTTGCCATGTTTTCTCCTATTGTGCCTTTTTGGATA
ACAGCAATCTATGTATTCTGTATTTCTCTGAAGCACTGCCATTGGTTCTT
TAACAACTATCTTAGGAAAAGAGTCATGTTTAATGGAGTTACATTTAGTA
CCTTCGAGGAGGCTGCTTTGTGTACCTTTTTGCTCAACAAGGAAATGTAC
CTAAAATTGCGTAGCGAGACACTGTTGCCACTTACACAGTATAACAGGTA
TCTTGCTCTATATAACAAGTACAAGTATTTCAGTGGAGCCTTAGATACTA
CCAGCTATCGTGAAGCAGCTTGCTGCCACTTAGCAAAGGCTCTAAATGAC
TTTAGCAACTCAGGTGCTGATGTTCTCTACCAACCACCACAGACATCAAT
CACTTCTGCTGTTCTGCAGAGTGGTTTTAGGAAAATGGCATTCCCGTCAG
GCAAAGTTGAAGGGTGCATGGTACAAGTAACCTGTGGAACTACAACTCTT
AATGGATTGTGGTTGGATGACACAGTATACTGTCCAAGACATGTCATTTG
CACAGCAGAAGACATGCTTAATCCTAACTATGAAGATCTGCTCATTCGCA
AATCCAACCATAGCTTTCTTGTTCAGGCTGGCAATGTTCAACTTCGTGTT
ATTGGCCATTCTATGCAAAATTGTCTGCTTAGGCTTAAAGTTGATACTTC
TAACCCTAAGACACCCAAGTATAAATTTGTCCGTATCCAACCTGGTCAAA
CATTTTCAGTTCTAGCATGCTACAATGGTTCACCATCTGGTGTTTATCAG
TGTGCCATGAGACCTAATCATACCATTAAAGGTTCTTTCCTTAATGGATC
ATGTGGTAGTGTTGGTTTTAACATTGATTATGATTGCGTGTCTTTCTGCT
ATATGCATCATATGGAGCTTCCAACAGGAGTACACGCTGGTACTGACTTA
GAAGGTAAATTCTATGGTCCATTTGTTGACAGACAAACTGCACAGGCTGC
AGGTACAGACACAACCATAACATTAAATGTTTTGGCATGGCTGTATGCTG
CTGTTATCAATGGTGATAGGTGGTTTCTTAATAGATTCACCACTACTTTG
AATGACTTTAACCTTGTGGCAATGAAGTACAACTATGAACCTTTGACACA
AGATCATGTTGACATATTGGGACCTCTTTCTGCTCAAACAGGAATTGCCG
TCTTAGATATGTGTGCTGCTTTGAAAGAGCTGCTGCAGAATGGTATGAAT
GGTCGTACTATCCTTGGTAGCACTATTTTAGAAGATGAGTTTACACCATT
TGATGTTGTTAGACAATGCTCTGGTGTTACCTTCCAAGGTAAGTTCAAGA
AAATTGTTAAGGGCACTCATCATTGGATGCTTTTAACTTTCTTGACATCA
CTATTGATTCTTGTTCAAAGTACACAGTGGTCACTGTTTTTCTTTGTTTA
CGAGAATGCTTTCTTGCCATTTACTCTTGGTATTATGGCAATTGCTGCAT
GTGCTATGCTGCTTGTTAAGCATAAGCACGCATTCTTGTGCTTGTTTCTG
TTACCTTCTCTTGCAACAGTTGCTTACTTTAATATGGTCTACATGCCTGC
TAGCTGGGTGATGCGTATCATGACATGGCTTGAATTGGCTGACACTAGCT
TGTCTGGTTATAGGCTTAAGGATTGTGTTATGTATGCTTCAGCTTTAGTT
TTGCTTATTCTCATGACAGCTCGCACTGTTTATGATGATGCTGCTAGACG
TGTTTGGACACTGATGAATGTCATTACACTTGTTTACAAAGTCTACTATG
GTAATGCTTTAGATCAAGCTATTTCCATGTGGGCCTTAGTTATTTCTGTA
ACCTCTAACTATTCTGGTGTCGTTACGACTATCATGTTTTTAGCTAGAGC
TATAGTGTTTGTGTGTGTTGAGTATTACCCATTGTTATTTATTACTGGCA
ACACCTTACAGTGTATCATGCTTGTTTATTGTTTCTTAGGCTATTGTTGC
TGCTGCTACTTTGGCCTTTTCTGTTTACTCAACCGTTACTTCAGGCTTAC
TCTTGGTGTTTATGACTACTTGGTCTCTACACAAGAATTTAGGTATATGA
ACTCCCAGGGGCTTTTGCCTCCTAAGAGTAGTATTGATGCTTTCAAGCTT
AACATTAAGTTGTTGGGTATTGGAGGTAAACCATGTATCAAGGTTGCTAC
TGTACAGTCTAAAATGTCTGACGTAAAGTGCACATCTGTGGTACTGCTCT
CGGTTCTTCAACAACTTAGAGTAGAGTCATCTTCTAAATTGTGGGCACAA
TGTGTACAACTCCACAATGATATTCTTCTTGCAAAAGACACAACTGAAGC
TTTCGAGAAGATGGTTTCTCTTTTGTCTGTTTTGCTATCCATGCAGGGTG
CTGTAGACATTAATAGGTTGTGCGAGGAAATGCTCGATAACCGTGCTACT
CTTCAGGCTATTGCTTCAGAATTTAGTTCTTTACCATCATATGCCGCTTA
TGCCACTGCCCAGGAGGCCTATGAGCAGGCTGTAGCTAATGGTGATTCTG
AAGTCGTTCTCAAAAAGTTAAAGAAATCTTTGAATGTGGCTAAATCTGAG
TTTGACCGTGATGCTGCCATGCAACGCAAGTTGGAAAAGATGGCAGATCA
GGCTATGACCCAAATGTACAAACAGGCAAGATCTGAGGACAAGAGGGCAA
AAGTAACTAGTGCTATGCAAACAATGCTCTTCACTATGCTTAGGAAGCTT
GATAATGATGCACTTAACAACATTATCAACAATGCGCGTGATGGTTGTGT
TCCACTCAACATCATACCATTGACTACAGCAGCCAAACTCATGGTTGTTG
TCCCTGATTATGGTACCTACAAGAACACTTGTGATGGTAACACCTTTACA
TATGCATCTGCACTCTGGGAAATCCAGCAAGTTGTTGATGCGGATAGCAA
GATTGTTCAACTTAGTGAAATTAACATGGACAATTCACCAAATTTGGCTT
GGCCTCTTATTGTTACAGCTCTAAGAGCCAACTCAGCTGTTAAACTACAG
AATAATGAACTGAGTCCAGTAGCACTACGACAGATGTCCTGTGCGGCTGG
TACCACACAAACAGCTTGTACTGATGACAATGCACTTGCCTACTATAACA
ATTCGAAGGGAGGTAGGTTTGTGCTGGCATTACTATCAGACCACCAAGAT
CTCAAATGGGCTAGATTCCCTAAGAGTGATGGTACAGGTACAATTTACAC
AGAACTGGAACCACCTTGTAGGTTTGTTACAGACACACCAAAAGGGCCTA
AAGTGAAATACTTGTACTTCATCAAAGGCTTAAACAACCTAAATAGAGGT
ATGGTGCTGGGCAGTTTAGCTGCTACAGTACGTCTTCAGGCTGGAAATGC
TACAGAAGTACCTGCCAATTCAACTGTGCTTTCCTTCTGTGCTTTTGCAG
TAGACCCTGCTAAAGCATATAAGGATTACCTAGCAAGTGGAGGACAACCA
ATCACCAACTGTGTGAAGATGTTGTGTACACACACTGGTACAGGACAGGC
AATTACTGTAACACCAGAAGCTAACATGGACCAAGAGTCCTTTGGTGGTG
CTTCATGTTGTCTGTATTGTAGATGCCACATTGACCATCCAAATCCTAAA
GGATTCTGTGACTTGAAAGGTAAGTACGTCCAAATACCTACCACTTGTGC
TAATGACCCAGTGGGTTTTACACTTAGAAACACAGTCTGTACCGTCTGCG
GAATGTGGAAAGGTTATGGCTGTAGTTGTGACCAACTCCGCGAACCCTTG
ATGCAGTCTGCGGATGCATCAACGTTTTTAAACGGGTTTGCGGTGTAAGT
GCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTCTA
CAGGGCTTTTGATATTTACAACGAAAAAGTTGCTGGTTTTGCAAAGTTCC
TAAAAACTAATTGCTGTCGCTTCCAGGAGAAGGATGAGGAAGGCAATTTA
TTAGACTCTTACTTTGTAGTTAAGAGGCATACTATGTCTAACTACCAACA
TGAAGAGACTATTTATAACTTGGTTAAAGATTGTCCAGCGGTTGCTGTCC
ATGACTTTTTCAAGTTTAGAGTAGATGGTGACATGGTACCACATATATCA
CGTCAGCGTCTAACTAAATACACAATGGCTGATTTAGTCTATGCTCTACG
TCATTTTGATGAGGGTAATTGTGATACATTAAAAGAAATACTCGTCACAT
ACAATTGCTGTGATGATGATTATTTCAATAAGAAGGATTGGTATGACTTC
GTAGAGAATCCTGACATCTTACGCGTATATGCTAACTTAGGTGAGCGTGT
ACGCCAATCATTATTAAAGACTGTACAATTCTGCGATGCTATGCGTGATG
CAGGCATTGTAGGCGTACTGACATTAGATAATCAGGATCTTAATGGGAAC
TGGTACGATTTCGGTGATTTCGTACAAGTAGCACCAGGCTGCGGAGTTCC
TATTGTGGATTCATATTACTCATTGCTGATGCCCATCCTCACTTTGACTA
GGGCATTGGCTGCTGAGTCCCATATGGATGCTGATCTCGCAAAACCACTT
ATTAAGTGGGATTTGCTGAAATATGATTTTACGGAAGAGAGACTTTGTCT
CTTCGACCGTTATTTTAAATATTGGGACCAGACATACCATCCCAATTGTA
TTAACTGTTTGGATGATAGGTGTATCCTTCATTGTGCAAACTTTAATGTG
TTATTTTCTACTGTGTTTCCACCTACAAGTTTTGGACCACTAGTAAGAAA
AATATTTGTAGATGGTGTTCCTTTTGTTGTTTCAACTGGATACCATTTTC
GTGAGTTAGGAGTCGTACATAATCAGGATGTAAACTTACATAGCTCGCGT
CTCAGTTTCAAGGAACTTTTAGTGTATGCTGCTGATCCAGCTATGCATGC
AGCTTCTGGCAATTTATTGCTAGATAAACGCACTACATGCTTTTCAGTAG
CTGCACTAACAAACAATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTT
AATAAAGACTTTTATGACTTTGCTGTGTCTAAAGGTTTCTTTAAGGAAGG
AAGTTCTGTTGAACTAAAACACTTCTTCTTTGCTCAGGATGGCAACGCTG
CTATCAGTGATTATGACTATTATCGTTATAATCTGCCAACAATGTGTGAT
ATCAGACAACTCCTATTCGTAGTTGAAGTTGTTGATAAATACTTTGATTG
TTACGATGGTGGCTGTATTAATGCCAACCAAGTAATCGTTAACAATCTGG
ATAAATCAGCTGGTTTCCCATTTAATAAATGGGGTAAGGCTAGACTTTAT
TATGACTCAATGAGTTATGAGGATCAAGATGCACTTTTCGCGTATACTAA
GCGTAATGTCATCCCTACTATAACTCAAATGAATCTTAAGTATGCCATTA
GTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACT
ATGACAAATAGACAGTTTCATCAGAAATTATTGAAGTCAATAGCCGCCAC
TAGAGGAGCTACTGTGGTAATTGGAACAAGCAAGTTTTACGGTGGCTGGC
ATAATATGTTAAAAACTGTTTACAGTGATGTAGAAACTCCACACCTTATG
GGTTGGGATTATCCAAAATGTGACAGAGCCATGCCTAACATGCTTAGGAT
AATGGCCTCTCTTGTTCTTGCTCGCAAACATAACACTTGCTGTAACTTAT
CACACCGTTTCTACAGGTTAGCTAACGAGTGTGCGCAAGTATTAAGTGAG
ATGGTCATGTGTGGCGGCTCACTATATGTTAAACCAGGTGGAACATCATC
CGGTGATGCTACAACTGCTTATGCTAATAGTGTCTTTAACATTTGTCAAG
CTGTTACAGCCAATGTAAATGCACTTCTTTCAACTGATGGTAATAAGATA
GCTGACAAGTATGTCCGCAATCTACAACACAGGCTCTATGAGTGTCTCTA
TAGAAATAGGGATGTTGATCATGAATTCGTGGATGAGTTTTACGCTTACC
TGCGTAAACATTTCTCCATGATGATTCTTTCTGATGATGCCGTTGTGTGC
TATAACAGTAACTATGCGGCTCAAGGTTTAGTAGCTAGCATTAAGAACTT
TAAGGCAGTTCTTTATTATCAAAATAATGTGTTCATGTCTGAGGCAAAAT
GTTGGACTGAGACTGACCTTACTAAAGGACCTCACGAATTTTGCTCACAG
CATACAATGCTAGTTAAACAAGGAGATGATTACGTGTACCTGCCTTACCC
AGATCCATCAAGAATATTAGGCGCAGGCTGTTTTGTCGATGATATTGTCA
AAACAGATGGTACACTTATGATTGAAAGGTTCGTGTCACTGGCTATTGAT
GCTTACCCACTTACAAAACATCCTAATCAGGAGTATGCTGATGTCTTTCA
CTTGTATTTACAATACATTAGAAAGTTACATGATGAGCTTACTGGCCACA
TGTTGGACATGTATTCCGTAATGCTAACTAATGATAACACCTCACGGTAC
TGGGAACCTGAGTTTTATGAGGCTATGTACACACCACATACAGTCTTGCA
GGCTGTAGGTGCTTGTGTATTGTGCAATTCACAGACTTCACTTCGTTGCG
GTGCCTGTATTAGGAGACCATTCCTATGTTGCAAGTGCTGCTATGACCAT
GTCATTTCAACATCACACAAATTAGTGTTGTCTGTTAATCCCTATGTTTG
CAATGCCCCAGGTTGTGATGTCACTGATGTGACACAACTGTATCTAGGAG
GTATGAGCTATTATTGCAAGTCACATAAGCCTCCCATTAGTTTTCCATTA
TGTGCTAATGGTCAGGTTTTTGGTTTATACAAAAACACATGTGTAGGCAG
TGACAATGTCACTGACTTCAATGCGATAGCAACATGTGATTGGACTAATG
CTGGCGATTACATACTTGCCAACACTTGTACTGAGAGACTCAAGCTTTTC
GCAGCAGAAACGCTCAAAGCCACTGAGGAAACATTTAAGCTGTCATATGG
TATTGCCACTGTACGCGAAGTACTCTCTGACAGAGAATTGCATCTTTCAT
GGGAGGTTGGAAAACCTAGACCACCATTGAACAGAAACTATGTCTTTACT
GGTTACCGTGTAACTAAAAATAGTAAAGTACAGATTGGAGAGTACACCTT
TGAAAAAGGTGACTATGGTGATGCTGTTGTGTACAGAGGTACTACGACAT
ACAAGTTGAATGTTGGTGATTACTTTGTGTTGACATCTCACACTGTAATG
CCACTTAGTGCACCTACTCTAGTGCCACAAGAGCACTATGTGAGAATTAC
TGGCTTGTACCCAACACTCAACATCTCAGATGAGTTTTCTAGCAATGTTG
CAAATTATCAAAAGGTCGGCATGCAAAAGTACTCTACACTCCAAGGACCA
CCTGGTACTGGTAAGAGTCATTTTGCCATCGGACTTGCTCTCTATTACCC
ATCTGCTCGCATAGTGTATACGGCATGCTCTCATGCAGCTGTTGATGCCC
TATGTGAAAAGGCATTAAAATATTTGCCCATAGATAAATGTAGTAGAATC
ATACCTGCGCGTGCGCGCGTAGAGTGTTTTGATAAATTCAAAGTGAATTC
AACACTAGAACAGTATGTTTTCTGCACTGTAAATGCATTGCCAGAAACAA
CTGCTGACATTGTAGTCTTTGATGAAATCTCTATGGCTACTAATTATGAC
TTGAGTGTTGTCAATGCTAGACTTCGTGCAAAACACTACGTCTATATTGG
CGATCCTGCTCAATTACCAGCCCCCCGCACATTGCTGACTAAAGGCACAC
TAGAACCAGAATATTTTAATTCAGTGTGCAGACTTATGAAAACAATAGGT
CCAGACATGTTCCTTGGAACTTGTCGCCGTTGTCCTGCTGAAATTGTTGA
CACTGTGAGTGCTTTAGTTTATGACAATAAGCTAAAAGCACACAAGGATA
AGTCAGCTCAATGCTTCAAAATGTTCTACAAAGGTGTTATTACACATGAT
GTTTCATCTGCAATCAACAGACCTCAAATAGGCGTTGTAAGAGAATTTCT
TACACGCAATCCTGCTTGGAGAAAAGCTGTTTTTATCTCACCTTATAATT
CACAGAACGCTGTAGCTTCAAAAATCTTAGGATTGCCTACGCAGACTGTT
GATTCATCACAGGGTTCTGAATATGACTATGTCATATTCACACAAACTAC
TGAAACAGCACACTCTTGTAATGTCAACCGCTTCAATGTGGCTATCACAA
GGGCAAAAATTGGCATTTTGTGCATAATGTCTGATAGAGATCTTTATGAC
AAACTGCAATTTACAAGTCTAGAAATACCACGTCGCAATGTGGCTACATT
ACAAGCAGAAAATGTAACTGGACTTTTTAAGGACTGTAGTAAGATCATTA
CTGGTCTTCATCCTACACAGGCACCTACACACCTCAGCGTTGATATAAAG
TTCAAGACTGAAGGATTATGTGTTGACATACCAGGCATACCAAAGGACAT
GACCTACCGTAGACTCATCTCTATGATGGGTTTCAAAATGAATTACCAAG
TCAATGGTTACCCTAATATGTTTATCACCCGCGAAGAAGCTATTCGTCAC
GTTCGTGCGTGGATTGGCTTTGATGTAGAGGGCTGTCATGCAACTAGAGA
TGCTGTGGGTACTAACCTACCTCTCCAGCTAGGATTTTCTACAGGTGTTA
ACTTAGTAGCTGTACCGACTGGTTATGTTGACACTGAAAATAACACAGAA
TTCACCAGAGTTAATGCAAAACCTCCACCAGGTGACCAGTTTAAACATCT
TATACCACTCATGTATAAAGGCTTGCCCTGGAATGTAGTGCGTATTAAGA
TAGTACAAATGCTCAGTGATACACTGAAAGGATTGTCAGACAGAGTCGTG
TTCGTCCTTTGGGCGCATGGCTTTGAGCTTACATCAATGAAGTACTTTGT
CAAGATTGGACCTGAAAGAACGTGTTGTCTGTGTGACAAACGTGCAACTT
GCTTTTCTACTTCATCAGATACTTATGCCTGCTGGAATCATTCTGTGGGT
TTTGACTATGTCTATAACCCATTTATGATTGATGTTCAGCAGTGGGGCTT
TACGGGTAACCTTCAGAGTAACCATGACCAACATTGCCAGGTACATGGAA
ATGCACATGTGGCTAGTTGTGATGCTATCATGACTAGATGTTTAGCAGTC
CATGAGTGCTTTGTTAAGCGCGTTGATTGGTCTGTTGAATACCCTATTAT
AGGAGATGAACTGAGGGTTAATTCTGCTTGCAGAAAAGTACAACACATGG
TTGTGAAGTCTGCATTGCTTGCTGATAAGTTTCCAGTTCTTCATGACATT
GGAAATCCAAAGGCTATCAAGTGTGTGCCTCAGGCTGAAGTAGAATGGAA
GTTCTACGATGCTCAGCCATGTAGTGACAAAGCTTACAAAATAGAGGAAC
TCTTCTATTCTTATGCTACACATCACGATAAATTCACTGATGGTGTTTGT
TTGTTTTGGAATTGTAACGTTGATCGTTACCCAGCCAATGCAATTGTGTG
TAGGTTTGACACAAGAGTCTTGTCAAACTTGAACTTACCAGGCTGTGATG
GTGGTAGTTTGTATGTGAATAAGCATGCATTCCACACTCCAGCTTTCGAT
AAAAGTGCATTTACTAATTTAAAGCAATTGCCTTTCTTTTACTATTCTGA
TAGTCCTTGTGAGTCTCATGGCAAACAAGTAGTGTCGGATATTGATTATG
TTCCACTCAAATCTGCTACGTGTATTACACGATGCAATTTAGGTGGTGCT
GTTTGCAGACACCATGCAAATGAGTACCGACAGTACTTGGATGCATATAA
TATGATGATTTCTGCTGGATTTAGCCTATGGATTTACAAACAATTTGATA
CTTATAACCTGTGGAATACATTTACCAGGTTACAGAGTTTAGAAAATGTG
GCTTATAATGTTGTTAATAAAGGACACTTTGATGGACACGCCGGCGAAGC
ACCTGTTTCCATCATTAATAATGCTGTTTACACAAAGGTAGATGGTATTG
ATGTGGAGATCTTTGAAAATAAGACAACACTTCCTGTTAATGTTGCATTT
GAGCTTTGGGCTAAGCGTAACATTAAACCAGTGCCAGAGATTAAGATACT
CAATAATTTGGGTGTTGATATCGCTGCTAATACTGTAATCTGGGACTACA
AAAGAGAAGCCCCAGCACATGTATCTACAATAGGTGTCTGCACAATGACT
GACATTGCCAAGAAACCTACTGAGAGTGCTTGTTCTTCACTTACTGTCTT
GTTTGATGGTAGAGTGGAAGGACAGGTAGACCTTTTTAGAAACGCCCGTA
ATGGTGTTTTAATAACAGAAGGTTCAGTCAAAGGTCTAACACCTTCAAAG
GGACCAGCACAAGCTAGCGTCAATGGAGTCACATTAATTGGAGAATCAGT
AAAAACACAGTTTAACTACTTTAAGAAAGTAGACGGCATTATTCAACAGT
TGCCTGAAACCTACTTTACTCAGAGCAGAGACTTAGAGGATTTTAAGCCC
AGATCACAAATGGAAACTGACTTTCTCGAGCTCGCTATGGATGAATTCAT
ACAGCGATATAAGCTCGAGGGCTATGCCTTCGAACACATCGTTTATGGAG
ATTTCAGTCATGGACAACTTGGCGGTCTTCATTTAATGATAGGCTTAGCC
AAGCGCTCACAAGATTCACCACTTAAATTAGAGGATTTTATCCCTATGGA
CAGCACAGTGAAAAATTACTTCATAACAGATGCGCAAACAGGTTCATCAA
AATGTGTGTGTTCTGTGATTGATCTTTTACTTGATGACTTTGTCGAGATA
ATAAAGTCACAAGATTTGTCAGTGATTTCAAAAGTGGTCAAGGTTACAAT
TGACTATGCTGAAATTTCATTCATGCTTTGGTGTAAGGATGGACATGTTG
AAACCTTCTACCCAAAACTACAAGCAAGTCGAGCGTGGCAACCAGGTGTT
GCGATGCCTAACTTGTACAAGATGCAAAGAATGCTTCTTGAAAAGTGTGA
CCTTCAGAATTATGGTGAAAATGCTGTTATACCAAAAGGAATAATGATGA
ATGTCGCAAAGTATACTCAACTGTGTCAATACTTAAATACACTTACTTTA
GCTGTACCCTACAACATGAGAGTTATTCACTTTGGTGCTGGCTCTGATAA
AGGAGTTGCACCAGGTACAGCTGTGCTCAGACAATGGTTGCCAACTGGCA
CACTACTTGTCGATTCAGATCTTAATGACTTCGTCTCCGACGCATATTCT
ACTTTAATTGGAGACTGTGCAACAGTACATACGGCTAATAAATGGGACCT
TATTATTAGCGATATGTATGACCCTAGGACCAAACATGTGACAAAAGAGA
ATGACTCTAAAGAAGGGTTTTTCACTTATCTGTGTGGATTTATAAAGCAA
AAACTAGCCCTGGGTGGTTCTATAGCTGTAAAGATAACAGAGCATTCTTG
GAATGCTGACCTTTACAAGCTTATGGGCCATTTCTCATGGTGGACAGCTT
TTGTTACAAATGTAAATGCATCATCATCGGAAGCATTTTTAATTGGGGCT
AACTATCTTGGCAAGCCGAAGGAACAAATTGATGGCTATACCATGCATGC
TAACTACATTTTCTGGAGGAACACAAATCCTATCCAGTTGTCTTCCTATT
CACTCTTTGACATGAGCAAATTTCCTCTTAAATTAAGAGGAACTGCTGTA
ATGTCTCTTAAGGAGAATCAAATCAATGATATGATTTATTCTCTTCTGGA
AAAAGGTAGGCTTATCATTAGAGAAAACAACAGAGTTGTGGTTTCAAGTG
ATATTCTTGTTAACAACTAAACGAACATGTTTATTTTCTTATTATTTCTT
ACTCTCACTAGTGGTAGTGACCTTGACCGGTGCACCACTTTTGATGATGT
TCAAGCTCCTAATTACACTCAACATACTTCATCTATGAGGGGGGTTTACT
ATCCTGATGAAATTTTTAGATCAGACACTCTTTATTTAACTCAGGATTTA
TTTCTTCCATTTTATTCTAATGTTACAGGGTTTCATACTATTAATCATAC
GTTTGGCAACCCTGTCATACCTTTTAAGGATGGTATTTATTTTGCTGCCA
CAGAGAAATCAAATGTTGTCCGTGGTTGGGTTTTTGGTTCTACCATGAAC
AACAAGTCACAGTCGGTGATTATTATTAACAATTCTACTAATGTTGTTAT
ACGAGCATGTAACTTTGAATTGTGTGACAACCCTTTCTTTGCTGTTTCTA
AACCCATGGGTACACAGACACATACTATGATATTCGATAATGCATTTAAT
TGCACTTTCGAGTACATATCTGATGCCTTTTCGCTTGATGTTTCAGAAAA
GTCAGGTAATTTTAAACACTTACGAGAGTTTGTGTTTAAAAATAAAGATG
GGTTTCTCTATGTTTATAAGGGCTATCAACCTATAGATGTAGTTCGTGAT
CTACCTTCTGGTTTTAACACTTTGAAACCTATTTTTAAGTTGCCTCTTGG
TATTAACATTACAAATTTTAGAGCCATTCTTACAGCCTTTTCACCTGCTC
AAGACATTTGGGGCACGTCAGCTGCAGCCTATTTTGTTGGCTATTTAAAG
CCAACTACATTTATGCTCAAGTATGATGAAAATGGTACAATCACAGATGC
TGTTGATTGTTCTCAAAATCCACTTGCTGAACTCAAATGCTCTGTTAAGA
GCTTTGAGATTGACAAAGGAATTTACCAGACCTCTAATTTCAGGGTTGTT
CCCTCAGGAGATGTTGTGAGATTCCCTAATATTACAAACTTGTGTCCTTT
TGGAGAGGTTTTTAATGCTACTAAATTCCCTTCTGTCTATGCATGGGAGA
GAAAAAAAATTTCTAATTGTGTTGCTGATTACTCTGTGCTCTACAACTCA
ACATTTTTTTCAACCTTTAAGTGCTATGGCGTTTCTGCCACTAAGTTGAA
TGATCTTTGCTTCTCCAATGTCTATGCAGATTCTTTTGTAGTCAAGGGAG
ATGATGTAAGACAAATAGCGCCAGGACAAACTGGTGTTATTGCTGATTAT
AATTATAAATTGCCAGATGATTTCATGGGTTGTGTCCTTGCTTGGAATAC
TAGGAACATTGATGCTACTTCAACTGGTAATTATAATTATAAATATAGGT
ATCTTAGACATGGCAAGCTTAGGCCCTTTGAGAGAGACATATCTAATGTG
CCTTTCTCCCCTGATGGCAAACCTTGCACCCCACCTGCTCTTAATTGTTA
TTGGCCATTAAATGATTATGGTTTTTACACCACTACTGGCATTGGCTACC
AACCTTACAGAGTTGTAGTACTTTCTTTTGAACTTTTAAATGCACCGGCC
ACGGTTTGTGGACCAAAATTATCCACTGACCTTATTAAGAACCAGTGTGT
CAATTTTAATTTTAATGGACTCACTGGTACTGGTGTGTTAACTCCTTCTT
CAAAGAGATTTCAACCATTTCAACAATTTGGCCGTGATGTTTCTGATTTC
ACTGATTCCGTTCGAGATCCTAAAACATCTGAAATATTAGACATTTCACC
TTGCGCTTTTGGGGGTGTAAGTGTAATTACACCTGGAACAAATGCTTCAT
CTGAAGTTGCTGTTCTATATCAAGATGTTAACTGCACTGATGTTTCTACA
GCAATTCATGCAGATCAACTCACACCAGCTTGGCGCATATATTCTACTGG
AAACAATGTATTCCAGACTCAAGCAGGCTGTCTTATAGGAGCTGAGCATG
TCGACACTTCTTATGAGTGCGACATTCCTATTGGAGCTGGCATTTGTGCT
AGTTACCATACAGTTTCTTTATTACGTAGTACTAGCCAAAAATCTATTGT
GGCTTATACTATGTCTTTAGGTGCTGATAGTTCAATTGCTTACTCTAATA
ACACCATTGCTATACCTACTAACTTTTCAATTAGCATTACTACAGAAGTA
ATGCCTGTTTCTATGGCTAAAACCTCCGTAGATTGTAATATGTACATCTG
CGGAGATTCTACTGAATGTGCTAATTTGCTTCTCCAATATGGTAGCTTTT
GCACACAACTAAATCGTGCACTCTCAGGTATTGCTGCTGAACAGGATCGC
AACACACGTGAAGTGTTCGCTCAAGTCAAACAAATGTACAAAACCCCAAC
TTTGAAATATTTTGGTGGTTTTAATTTTTCACAAATATTACCTGACCCTC
TAAAGCCAACTAAGAGGTCTTTTATTGAGGACTTGCTCTTTAATAAGGTG
ACACTCGCTGATGCTGGCTTCATGAAGCAATATGGCGAATGCCTAGGTGA
TATTAATGCTAGAGATCTCATTTGTGCGCAGAAGTTCAATGGACTTACAG
TGTTGCCACCTCTGCTCACTGATGATATGATTGCTGCCTACACTGCTGCT
CTAGTTAGTGGTACTGCCACTGCTGGATGGACATTTGGTGCTGGCGCTGC
TCTTCAAATACCTTTTGCTATGCAAATGGCATATAGGTTCAATGGCATTG
GAGTTACCCAAAATGTTCTCTATGAGAACCAAAAACAAATCGCCAACCAA
TTTAACAAGGCGATTAGTCAAATTCAAGAATCACTTACAACAACATCAAC
TGCATTGGGCAAGCTGCAAGACGTTGTTAACCAGAATGCTCAAGCATTAA
ACACACTTGTTAAACAACTTAGCTCTAATTTTGGTGCAATTTCAAGTGTG
CTAAATGATATCCTTTCGCGACTTGATAAAGTCGAGGCGGAGGTACAAAT
TGACAGGTTAATTACAGGCAGACTTCAAAGCCTTCAAACCTATGTAACAC
AACAACTAATCAGGGCTGCTGAAATCAGGGCTTCTGCTAATCTTGCTGCT
ACTAAAATGTCTGAGTGTGTTCTTGGACAATCAAAAAGAGTTGACTTTTG
TGGAAAGGGCTACCACCTTATGTCCTTCCCACAAGCAGCCCCGCATGGTG
TTGTCTTCCTACATGTCACGTATGTGCCATCCCAGGAGAGGAACTTCACC
ACAGCGCCAGCAATTTGTCATGAAGGCAAAGCATACTTCCCTCGTGAAGG
TGTTTTTGTGTTTAATGGCACTTCTTGGTTTATTACACAGAGGAACTTCT
TTTCTCCACAAATAATTACTACAGACAATACATTTGTCTCAGGAAATTGT
GATGTCGTTATTGGCATCATTAACAACACAGTTTATGATCCTCTGCAACC
TGAGCTTGACTCATTCAAAGAAGAGCTGGACAAGTACTTCAAAAATCATA
CATCACCAGATGTTGATCTTGGCGACATTTCAGGCATTAACGCTTCTGTC
GTCAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTCGCTAAAAATTT
AAATGAATCACTCATTGACCTTCAAGAATTGGGAAAATATGAGCAATATA
TTAAATGGCCTTGGTATGTTTGGCTCGGCTTCATTGCTGGACTAATTGCC
ATCGTCATGGTTACAATCTTGCTTTGTTGCATGACTAGTTGTTGCAGTTG
CCTCAAGGGTGCATGCTCTTGTGGTTCTTGCTGCAAGTTTGATGAGGATG
ACTCTGAGCCAGTTCTCAAGGGTGTCAAATTACATTACACATAAACGAAC
TTATGGATTTGTTTATGAGATTTTTTACTCTTGGATCAATTACTGCACAG
CCAGTAAAAATTGACAATGCTTCTCCTGCAAGTACTGTTCATGCTACAGC
AACGATACCGCTACAAGCCTCACTCCCTTTCGGATGGCTTGTTATTGGCG
TTGCATTTCTTGCTGTTTTTCAGAGCGCTACCAAAATAATTGCGCTCAAT
AAAAGATGGCAGCTAGCCCTTTATAAGGGCTTCCAGTTCATTTGCAATTT
ACTGCTGCTATTTGTTACCATCTATTCACATCTTTTGCTTGTCGCTGCAG
GTATGGAGGCGCAATTTTTGTACCTCTATGCCTTGATATATTTTCTACAA
TGCATCAACGCATGTAGAATTATTATGAGATGTTGGCTTTGTTGGAAGTG
CAAATCCAAGAACCCATTACTTTATGATGCCAACTACTTTGTTTGCTGGC
ACACACATAACTATGACTACTGTATACCATATAACAGTGTCACAGATACA
ATTGTCGTTACTGAAGGTGACGGCATTTCAACACCAAAACTCAAAGAAGA
CTACCAAATTGGTGGTTATTCTGAGGATAGGCACTCAGGTGTTAAAGACT
ATGTCGTTGTACATGGCTATTTCACCGAAGTTTACTACCAGCTTGAGTCT
ACACAAATTACTACAGACACTGGTATTGAAAATGCTACATTCTTCATCTT
TAACAAGCTTGTTAAAGACCCACCGAATGTGCAAATACACACAATCGACG
GCTCTTCAGGAGTTGCTAATCCAGCAATGGATCCAATTTATGATGAGCCG
ACGACGACTACTAGCGTGCCTTTGTAAGCACAAGAAAGTGAGTACGAACT
TATGTACTCATTCGTTTCGGAAGAAACAGGTACGTTAATAGTTAATAGCG
TACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTCACACTAGCCATC
CTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAG
TTTAGTAAAACCAACGGTTTACGTCTACTCGCGTGTTAAAAATCTGAACT
CTTCTGAAGGAGTTCCTGATCTTCTGGTCTAAACGAACTAACTATTATTA
TTATTCTGTTTGGAACTTTAACATTGCTTATCATGGCAGACAACGGTACT
ATTACCGTTGAGGAGCTTAAACAACTCCTGGAACAATGGAACCTAGTAAT
AGGTTTCCTATTCCTAGCCTGGATTATGTTACTACAATTTGCCTATTCTA
ATCGGAACAGGTTTTTGTACATAATAAAGCTTGTTTTCCTCTGGCTCTTG
TGGCCAGTAACACTTGCTTGTTTTGTGCTTGCTGCTGTCTACAGAATTAA
TTGGGTGACTGGCGGGATTGCGATTGCAATGGCTTGTATTGTAGGCTTGA
TGTGGCTTAGCTACTTCGTTGCTTCCTTCAGGCTGTTTGCTCGTACCCGC
TCAATGTGGTCATTCAACCCAGAAACAAACATTCTTCTCAATGTGCCTCT
CCGGGGGACAATTGTGACCAGACCGCTCATGGAAAGTGAACTTGTCATTG
GTGCTGTGATCATTCGTGGTCACTTGCGAATGGCCGGACACTCCCTAGGG
CGCTGTGACATTAAGGACCTGCCAAAAGAGATCACTGTGGCTACATCACG
AACGCTTTCTTATTACAAATTAGGAGCGTCGCAGCGTGTAGGCACTGATT
CAGGTTTTGCTGCATACAACCGCTACCGTATTGGAAACTATAAATTAAAT
ACAGACCACGCCGGTAGCAACGACAATATTGCTTTGCTAGTACAGTAAGT
GACAACAGATGTTTCATCTTGTTGACTTCCAGGTTACAATAGCAGAGATA
TTGATTATCATTATGAGGACTTTCAGGATTGCTATTTGGAATCTTGACGT
TATAATAAGTTCAATAGTGAGACAATTATTTAAGCCTCTAACTAAGAAGA
ATTATTCGGAGTTAGATGATGAAGAACCTATGGAGTTAGATTATCCATAA
AACGAACATGAAAATTATTCTCTTCCTGACATTGATTGTATTTACATCTT
GCGAGCTATATCACTATCAGGAGTGTGTTAGAGGTACGACTGTACTACTA
AAAGAACCTTGCCCATCAGGAACATACGAGGGCAATTCACCATTTCACCC
TCTTGCTGACAATAAATTTGCACTAACTTGCACTAGCACACACTTTGCTT
TTGCTTGTGCTGACGGTACTCGACATACCTATCAGCTGCGTGCAAGATCA
GTTTCACCAAAACTTTTCATCAGACAAGAGGAGGTTCAACAAGAGCTCTA
CTCGCCACTTTTTCTCATTGTTGCTGCTCTAGTATTTTTAATACTTTGCT
TCACCATTAAGAGAAAGACAGAATGAATGAGCTCACTTTAATTGACTTCT
ATTTGTGCTTTTTAGCCTTTCTGCTATTCCTTGTTTTAATAATGCTTATT
ATATTTTGGTTTTCACTCGAAATCCAGGATCTAGAAGAACCTTGTACCAA
AGTCTAAACGAACATGAAACTTCTCATTGTTTTGACTTGTATTTCTCTAT
GCAGTTGCATATGCACTGTAGTACAGCGCTGTGCATCTAATAAACCTCAT
GTGCTTGAAGATCCTTGTAAGGTACAACACTAGGGGTAATACTTATAGCA
CTGCTTGGCTTTGTGCTCTAGGAAAGGTTTTACCTTTTCATAGATGGCAC
ACTATGGTTCAAACATGCACACCTAATGTTACTATCAACTGTCAAGATCC
AGCTGGTGGTGCGCTTATAGCTAGGTGTTGGTACCTTCATGAAGGTCACC
AAACTGCTGCATTTAGAGACGTACTTGTTGTTTTAAATAAACGAACAAAT
TAAAATGTCTGATAATGGACCCCAATCAAACCAACGTAGTGCCCCCCGCA
TTACATTTGGTGGACCCACAGATTCAACTGACAATAACCAGAATGGAGGA
CGCAATGGGGCAAGGCCAAAACAGCGCCGACCCCAAGGTTTACCCAATAA
TACTGCGTCTTGGTTCACAGCTCTCACTCAGCATGGCAAGGAGGAACTTA
GATTCCCTCGAGGCCAGGGCGTTCCAATCAACACCAATAGTGGTCCAGAT
GACCAAATTGGCTACTACCGAAGAGCTACCCGACGAGTTCGTGGTGGTGA
CGGCAAAATGAAAGAGCTCAGCCCCAGATGGTACTTCTATTACCTAGGAA
CTGGCCCAGAAGCTTCACTTCCCTACGGCGCTAACAAAGAAGGCATCGTA
TGGGTTGCAACTGAGGGAGCCTTGAATACACCCAAAGACCACATTGGCAC
CCGCAATCCTAATAACAATGCTGCCACCGTGCTACAACTTCCTCAAGGAA
CAACATTGCCAAAAGGCTTCTACGCAGAGGGAAGCAGAGGCGGCAGTCAA
GCCTCTTCTCGCTCCTCATCACGTAGTCGCGGTAATTCAAGAAATTCAAC
TCCTGGCAGCAGTAGGGGAAATTCTCCTGCTCGAATGGCTAGCGGAGGTG
GTGAAACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACCAGCTTGAG
AGCAAAGTTTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAA
GAAATCTGCTGCTGAGGCATCTAAAAAGCCTCGCCAAAAACGTACTGCCA
CAAAACAGTACAACGTCACTCAAGCATTTGGGAGACGTGGTCCAGAACAA
ACCCAAGGAAATTTCGGGGACCAAGACCTAATCAGACAAGGAACTGATTA
CAAACATTGGCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCT
TTGGAATGTCACGCATTGGCATGGAAGTCACACCTTCGGGAACATGGCTG
ACTTATCATGGAGCCATTAAATTGGATGACAAAGATCCACAATTCAAAGA
CAACGTCATACTGCTGAACAAGCACATTGACGCATACAAAACATTCCCAC
CAACAGAGCCTAAAAAGGACAAAAAGAAAAAGACTGATGAAGCTCAGCCT
TTGCCGCAGAGACAAAAGAAGCAGCCCACTGTGACTCTTCTTCCTGCGGC
TGACATGGATGATTTCTCCAGACAACTTCAAAATTCCATGAGTGGAGCTT
CTGCTGATTCAACTCAGGCATAAACACTCATGATGACCACACAAGGCAGA
TGGGCTATGTAAACGTTTTCGCAATTCCGTTTACGATACATAGTCTACTC
TTGTGCAGAATGAATTCTCGTAACTAAACAGCACAAGTAGGTTTAGTTAA
CTTTAATCTCACATAGCAATCTTTAATCAATGTGTAACATTAGGGAGGAC
TTGAAAGAGCCACCACATTTTCATCGAGGCCACGCGGAGTACGATCGAGG
GTACAGTGAATAATGCTAGGGAGAGCTGCCTATATGGAAGAGCCCTAATG
TGTAAAATTAATTTTAGTAGTGCTATCCCCATGTGATTTTAATAGCTTCT
TAGGAGAATGACAAAAAAAAAAAAAAAAAAAAAAAA
4
Human Genome Project
5
(No Transcript)
6
A Comparison of Genome Sets
Ensembl(29691)
Celera (39114)
6552
15231
9315
9300
747
604
364
Refseq (11015)
7
Sanger Method

• 1. A single strand of DNA to be sequenced
  (yellow) is hybridized to a 5 end labeled
  synthetic deoxynucleotide primer(Brown).

2. The primer is elongated using DNA polymerase
in four separate reaction mixtures
containing four normal deoxynucleotide
triphosphates (dNTPs) plus one of
dideoxynucleotide triphosphate (ddNTPs) in a
ratio of 100 1.
8
(No Transcript)
9
3. In each tube, the primer enlongation is
terminated by the incorporation of a
dideoxynucleotide triphosphate into the newly
synthesized chain.
10
4. The synthesized DNA chains can then be
separated by gel electrophoresis. Using this gel
analysis of fragments, the sequence of the
template DNA chain be determined.
11
New Sequencing Methods
1. Sequencing by MALDI-TOF Mass Spectrometry
2. Sequencing by Hybridization
3. Pyrosequencing
4. Atomic-Force Microscopy
5. Single-Molecule Fluorescence Microscopy
6. Nanopore Sequencing
12
Sequencing by MALDI-TOF Mass Spectrometry
MALDI was first coupled to time-of-flight mass
spectrometry in 1988 by Karas Hillenkamp. Their
remarkable innovation was that virtually any
macromolecule could be desorbed as an intact
gas-phase ion by embedding it in the crystal of a
low-molecular-weight molecule that strongly
absorbs energy from a pulse of laser light. Prior
to this landmark paper, laser desorption mass
spectrometry was limited to peptides with
specific volatility or photo-absorption
properties Although originally applied to
analysis of protein samples, MALDI-TOF-MS is now
widely used for oligonucleotides and DNAas well.
The essential features of MALDI-TOF-MS DNA
analysis are summarized as follows. The DNA
sample is typically dried at room temperature on
a flat surface in a matrix of 3-hydroxypicolic
acid. The 3-hydroxypicolic acid matrix serves the
critical purpose of absorbing UV light while
interacting very little with DNA. The sample is
then treated with a short pulse of UV laser light
that is absorbed by the 3-hydroxypicolic acid,
causing ablation of DNA ions into the gas phase.
The DNA ions are generally monovalent and intact.
After a specified time delay, the charged
gas-phase DNA molecules are extracted by a
high-voltage pulse and accelerated in an electric
field so that they attain a common kinetic
energy. They are subsequently passed into a
flight tube approximately 1 m long. Under vacuum
at a common kinetic energy, the relative time
required for a given molecule to travel the
flight path is dependent on its mass. At the end
of the flight tube, the molecules collide with an
ion-to-electron conversion detector, thus
registering the TOF from the original laser pulse
(t). The mass of a given analyte can then be
calculated from the relationship m 2qVt2 /2l2.
In practice, internal standards are often relied
on to confirm peak identities.
13
The electrospray process
14
The soft laser desorption process
15
Sequencing by MALDI-TOF Mass Spectrometry
MW spectrum of 33-mer 5-ACT AAT GGC AGT TCA TTG
CAT GAA TTT TAA AAG-3
16
DNA Sequencing by Hybridization
One early rationale for developing hybridization
arrays was de novo sequencing. As originally
conceived, this strategy sequencing by
hybridization (SBH) involved annealing a labeled
unknown DNA fragment to a complete array of short
oligonucleotides (e.g. all 65,336 combinations of
8-mers) and deciphering the unknown sequence from
the annealing pattern. Over the past decade, SBH
has largely been eclipsed by the use of DNA
arrays for single nucleotide polymorphisms (SNP)
and expression analysis. This is partly due to
the amount of diagnostic or biological
information to be gained per feature on the
array. For example, expression monitoring of the
entire human genome could be performed using a
microarray composed of 100,000 gene-specific
sequences (or possibly many fewer), whereas the
same number of features would allow resequencing
of only 25,000 bases. Another stumbling block for
sequencing applications is the use of short
oligonucleotide probes. These present such
problems as ambiguous reads as-sociated with
repeat regions within the unknown target
sequence, formation of secondary structures in
some oligonucleotide probes that result in little
or no detectable signal, and hybridization of
oligonucleotides with single mismatches (false
positives), which can be especially common at the
terminal base pair.
17
DNA Sequencing by Hybridization
This strategy involved annealing a labeled
unknown DNA fragment to a complete array of
short oligonucleotides (e.g. all 65,336
combinations of 8-mers) and deciphering the
unknown sequence from the annealing pattern.
18
Principle of Pyrosequencing
(http//www.pyrosequencing.com/pages/technology.ht
ml)
Pyrosequencing is to sequence DNA by enzymatic
DNA synthesis, and the DNA sequence is determined
the from the signal peak of released photons
during the synthesis. It includes the following 5
steps
Step 1A sequencing primer is hybridized to a
single stranded, PCR amplified, DNA template, and
incubated with the enzymes, DNA polymerase, ATP
sulfurylase, luciferase and apyrase, and the
substrates, adenosine 5 phosphosulfate (APS) and
luciferin.
                                                
                                 
Step 2The first of four deoxynucleotide
triphosphates (dNTP) is added to the reaction.
DNA polymerase catalyzes the incorporation of the
deoxynucleotide triphosphate into the DNA strand,
if it is complementary to the base in the
template strand. Each incorporation event is
accompanied by release of pyrophosphate (PPi) in
a quantity equimolar to the amount of
incorporated nucleotide.
                                               
                                  
                                               
                                  
19
Step 3ATP sulfurylase quantitatively converts
PPi to ATP in the presence of adenosine 5
phosphosulfate. This ATP drives the
luciferase-mediated conversion of luciferin to
oxyluciferin that generates visible light in
amounts that are proportional to the amount of
ATP. The light produced in the luciferase-catalyze
d reaction is detected by a charge coupled device
(CCD) camera and seen as a peak in a pyrogram.
Each light signal is proportional to the number
of nucleotides incorporated.
                                                
                                 
20
Step 4Apyrase, a nucleotide degrading enzyme,
continuously degrades unincorporated dNTPs and
excess ATP. When degradation is complete, another
dNTP is added.
Step 5Addition of dNTPs is performed one at a
time. It should be noted that deoxyadenosine
alfa-thio triphosphate (dATPaS) is used as a
substitute for the natural deoxyadenosine
triphosphate (dATP) since it is efficiently used
by the DNA polymerase, but not recognized by the
luciferase. As the process continues, the
complementary DNA strand is built up and the
nucleotide sequence is determined from the signal
peak in the pyrogram.
21
Summary of Pyrosequencing
Pyrosequencing is to sequence DNA by enzymatic
DNA synthesis, and the DNA sequence is
determined the from the signal peak of released
photons during the synthesis.
22
Zipper-sequencing of DNA
A DNA construct was engineered such that one of
its extremities had one strand anchored to a
surface via a long DNA fragment, and the other
strand was bound to a small bead, itself stuck to
a flexible glass fiber used as a force sensor.
As the surface is displaced, the molecule is
unzipped and the force to unpair two bases
measured by the force sensor.
23
Nanopore Sequencing of Polynucleotides
An interesting idea in sequencing DNA proposed by
D. Branton is to monitor the variation of ionic
current due to an applied electric field which
drives single-stranded polynucleotides through a
nanopore in a thin film. Preliminary results of
this method have shown its capability to
distinguish long stretches of the same
nucleotides, such as 30 adenines followed by 70
cytosines. Although this single molecule
sequencing method provides a great advantage to
sequence a long DNA, detection of monovalent ion
current through the a-hemolysin pore is not
likely to yield DNA sequence at single-nucleotide
resolution. . First, the translocation time
through the pore for each nucleotide is 1
microsecond, which is too short to resolve.
Second, the thermal fluctuation of translocation
time will forbid the possibility to determine the
number of repeat nucleotides for each blockade
current segment. Finally, the narrowest portion
of the channel pore is 50 Å long, meaning that
approximately seven nucleotides occupy that space
at a given instant. Each of those seven
nucleotides would contribute to resistance
against ionic current, thus obscuring the
influence of any single nucleotide.
24
Nanopore Sequencing of Polynucleotides
25
Sequencing DNA with a Rotating Field
26
The bond fluctuation model
Moving probability of each nucleic acid w
min1, exp(-DU/kT)
27
Translocation of DNA with Time
28
Translocation Time versus Frequency
29
Quantization of Translocation Time
30
Off-lattice simulations
31
Translocation Time versus Amplitude
32
Time Series of DNA Translocation
AAAAAAAAAAACGTACTTCGCGTGTAGTCATTTAATCCACCCCCCCCCCC
33
Prediction Error in Sequencing
AAAAAAAAAAAC (GTACTTCGCGTGTAGTCATTTAATCC)
ACCCCCCCCCCC
34
Fabricating Nanopore by Ion Beam
35
A Sequencing Array
36
Conclusions
1. The traditional Sanger method of sequencing
DNA is slow, costly, and inaccurate. It takes
about 15 years to sequence human DNA and costs
about 3 billion US dollars. The overlap of
predicted novel gene sets between Celera and
Ensembled is about 20.
2. The new method by nanopore sequencing can be
fast, inexpensive, and accurate. It takes about 1
day to sequence 100 million bases by using a
sequencing array and high accuracy can be
achieved by analyzing several time series.
3. The translocation time of polynucleotide
chains is well controlled by the frequency of the
rotating electric field. Specifically, it
increases linearly with the rotating period for
frequency less than 10 KHz.
4. The translocation time of each nucleotide is
quantized in unit of a quarter of rotating
period, which can be used to predict the sequence
accurately.