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 HIRA BATOOL
 MAIRA KHAN
 MARYAM ZAHRAH
 NADIA ASHRAF
NEXT
GENERATION
SEQUENCING
   Determining the sequence of the nucleotides i.e. A,T,C,G
    along a DNA strand
   Why DNA to be sequenced…???
   To know the order of nucleotides which will enable us to:
    › To know about genes
    › Find information about the architecture of the genome
    › Comparative genome analysis
 Plus-minus strand sequencing
 Maxam-Gilbert chemical sequencing
 Sanger`s chain termination sequencing (Dye
  termination sequencing)
      The most used method for last 30 years!
   By the passage of time new techniques were
    developed which replaced old techniques
    because they were:
    › Old
    › Time consuming
    › Laborious
    › Expensive
    › Hazardous(used hazardous reagents)
    › Volume of reagents and space
   Roche’s (454) GS FLX Genome Analyzer
   Illuminas Solexa 1G sequencer
   Applied Biosystem’s SOLiD system
   Helicos
 A DNA sequence that initially binds the RNA
  polymerase.
 Upstream to the transcription start site.
 Core promoter refers to the minimal set of
  sequence elements required for accurate
  transcription Initiation.
 Usually -35 to +35
 - 37 TO -32
 Consensus sequence: G/C G/C G/A CGCCC
 Recognized by TFIIB.
 The TFIIB–BRE interaction facilitates the
  assembly of a TFIIB–TBP–TATA complex
 -31 to – 26
 Consensus sequence : T A T A A/T A A/T
 Recognized by TBP( a subunit of TFIID)
 In Humans, 32% of 1031 potential promoter
  regions have one.
 Primary role is formation of pre-initiation
  complex(promoters + General TF).
 -2 TO +4
 C/T C/T A+1 N T/A C/T C/T
 Recognized by TFIID
 Nucleates PIC formation in TATA less
  promoter
 facilitates the binding of Transcription
  Factor II D (TBP)..
 + 28 to +32
 A/G G A/T CGTG
 Recognized by TFIID
 DPE plays a major role at TATA-less
  promoters.
 +18 TO +29
 Consensus sequence: CSARSSAACGC
 cooperate with the initiator to stimulate
  transcription.
 NO TATA in these promoters.
 DNA sequences in which Four guanine bases
  can associate through Hoogsteen hydrogen
  bonding to form a square planar structure
  called a G-tetrad, and two or more G- tetrads
  can stack on top of each other to form a G-
  quadruplex.
 Repeats of at least 3 guanine residues are
  separated by loops of 1-7 other base pairs
 Present in DNA, RNA, LNA (locked), PNA
  (peptide)
 Across a wide range of species, G4 DNA motifs
  were found in telomeres, G-rich micro- and
  mini-satellites, near promoters, and within the
  ribosomal DNA (rDNA)
 Important components of human telomeres,
  and play a role in regulation of transcription
  and translation.
 They are also interesting as nanotechnological
  devices..
 Generally, a simple pattern match is used for
  searching for possible quadruplex forming
  sequences:
 G3+N1-7G3+N1-7G3+N1-7G3+
                      where N is any base (including G)
.
   Text = Promoter or Telomere sequence
   Pattern= GGG
   N=text.length( )
   M=pattern.length( )
   Count=0                       [ count the no of G’s]
   Array[i][j]             [ stores start and end position]
   i=0                     [stores start]
   For(t=0;t<=N;)                                    [scans Text]
    {
           for(p=0;p<=M;)                                  [scans Pattern]
          {
               if(text[t]==Pattern[p])
                {
                        p++;
                        t++;
                         count++;
                }
               else
                      t++; count=0
if(count>=M)
         {
              j=0;
             array[i][j]=t -2;              [stores start]
             array[i][j+1]=t;               [stores end]
             i++;
          }
        t= prefix function( )
         if(arr[i][j] – arr[i-1][j+1] >0 && arr[i][j]- arr[i-
    1][j+1]!<=7)
             {
                  t= prefix function( )
              }
     }
}
   http://www.ploscompbiol.org/article/info%3
    Adoi%2F10.1371%2Fjournal.pcbi.1000861

 http://www.ncbi.nlm.nih.gov/pmc/articles/P
  MC1636468/
 Quadruplex.org
   R   Purine (A or G)
   Y   Pyrimidine (C or T)
   N   Any nucleotide
   W   Weak (A or T)
   S   Strong (G or C)
   M   Amino (A or C)
   K   Keto (G or T)
   B   Not A (G or C or T)
   H   Not G (A or C or T)
   D   Not C (A or G or T)
   V   Not T (A or G or C)
 PNA's backbone is composed of repeating N-
  (2-aminoethyl)-glycine units linked by peptide
  bonds. The bases are linked to the backbone
  by methylene carbonyl bonds.
 LNA The ribose moiety of an LNA nucleotide is
  modified with an extra bridge connecting the
  2' oxygen and 4' carbon

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Next generation sequencing

  • 1.  HIRA BATOOL  MAIRA KHAN  MARYAM ZAHRAH  NADIA ASHRAF
  • 2.
  • 4. Determining the sequence of the nucleotides i.e. A,T,C,G along a DNA strand  Why DNA to be sequenced…???  To know the order of nucleotides which will enable us to: › To know about genes › Find information about the architecture of the genome › Comparative genome analysis
  • 5.  Plus-minus strand sequencing  Maxam-Gilbert chemical sequencing  Sanger`s chain termination sequencing (Dye termination sequencing)  The most used method for last 30 years!
  • 6. By the passage of time new techniques were developed which replaced old techniques because they were: › Old › Time consuming › Laborious › Expensive › Hazardous(used hazardous reagents) › Volume of reagents and space
  • 7. Roche’s (454) GS FLX Genome Analyzer
  • 8. Illuminas Solexa 1G sequencer
  • 9. Applied Biosystem’s SOLiD system
  • 10. Helicos
  • 11.
  • 12.  A DNA sequence that initially binds the RNA polymerase.  Upstream to the transcription start site.  Core promoter refers to the minimal set of sequence elements required for accurate transcription Initiation.  Usually -35 to +35
  • 13.
  • 14.
  • 15.  - 37 TO -32  Consensus sequence: G/C G/C G/A CGCCC  Recognized by TFIIB.  The TFIIB–BRE interaction facilitates the assembly of a TFIIB–TBP–TATA complex
  • 16.  -31 to – 26  Consensus sequence : T A T A A/T A A/T  Recognized by TBP( a subunit of TFIID)  In Humans, 32% of 1031 potential promoter regions have one.  Primary role is formation of pre-initiation complex(promoters + General TF).
  • 17.  -2 TO +4  C/T C/T A+1 N T/A C/T C/T  Recognized by TFIID  Nucleates PIC formation in TATA less promoter  facilitates the binding of Transcription Factor II D (TBP)..
  • 18.  + 28 to +32  A/G G A/T CGTG  Recognized by TFIID  DPE plays a major role at TATA-less promoters.
  • 19.  +18 TO +29  Consensus sequence: CSARSSAACGC  cooperate with the initiator to stimulate transcription.  NO TATA in these promoters.
  • 20.
  • 21.  DNA sequences in which Four guanine bases can associate through Hoogsteen hydrogen bonding to form a square planar structure called a G-tetrad, and two or more G- tetrads can stack on top of each other to form a G- quadruplex.  Repeats of at least 3 guanine residues are separated by loops of 1-7 other base pairs
  • 22.
  • 23.  Present in DNA, RNA, LNA (locked), PNA (peptide)  Across a wide range of species, G4 DNA motifs were found in telomeres, G-rich micro- and mini-satellites, near promoters, and within the ribosomal DNA (rDNA)
  • 24.  Important components of human telomeres, and play a role in regulation of transcription and translation.  They are also interesting as nanotechnological devices..
  • 25.
  • 26.
  • 27.  Generally, a simple pattern match is used for searching for possible quadruplex forming sequences:  G3+N1-7G3+N1-7G3+N1-7G3+ where N is any base (including G) .
  • 28. Text = Promoter or Telomere sequence  Pattern= GGG  N=text.length( )  M=pattern.length( )  Count=0 [ count the no of G’s]  Array[i][j] [ stores start and end position]  i=0 [stores start]  For(t=0;t<=N;) [scans Text] { for(p=0;p<=M;) [scans Pattern] { if(text[t]==Pattern[p]) { p++; t++; count++; } else t++; count=0
  • 29. if(count>=M) { j=0; array[i][j]=t -2; [stores start] array[i][j+1]=t; [stores end] i++; } t= prefix function( ) if(arr[i][j] – arr[i-1][j+1] >0 && arr[i][j]- arr[i- 1][j+1]!<=7) { t= prefix function( ) } } }
  • 30.
  • 31. http://www.ploscompbiol.org/article/info%3 Adoi%2F10.1371%2Fjournal.pcbi.1000861  http://www.ncbi.nlm.nih.gov/pmc/articles/P MC1636468/  Quadruplex.org
  • 32. R Purine (A or G)  Y Pyrimidine (C or T)  N Any nucleotide  W Weak (A or T)  S Strong (G or C)  M Amino (A or C)  K Keto (G or T)  B Not A (G or C or T)  H Not G (A or C or T)  D Not C (A or G or T)  V Not T (A or G or C)
  • 33.  PNA's backbone is composed of repeating N- (2-aminoethyl)-glycine units linked by peptide bonds. The bases are linked to the backbone by methylene carbonyl bonds.  LNA The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon