Primer Design for PCR Amplification and Sequencing Reaction

 
 Extended Hyper-variable Segment I (15919 – 16569)
  |15919     16030|
5’-TGTAAACCGGAGATGAAAACCTTTTTCCAAGGACAAATCAGAGAAAAAGTCTTTAACTCCACCATTAGCACCCAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTC-3’
3’-ACATTTGGCCTCTACTTTTGGAAAAAGGTTCCTGTTTAGTCTCTTTTTCAGAAATTGAGGTGGTAATCGTGGGTTTCGATTCTAAGATTAAATTTGATAAGAGACAAGAAAG-5’
  |1 5’-TAACTCCACCATTAGCACCC-3’--> 112|
  |16031 16142|
5’-ATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATAC-3’
3’-TACCCCTTCGTCTAAACCCATGGTGGGTTCATAACTGAGTGGGTAGTTGTTGGCGATACATAAAGCATGTAATGACGGTCGGTGGTACTTATAACATGCCATGGTATTTATG-5’
  |113  224|
  |16143 16240|
5’-TTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAA--C----CCCCT-C-C-CC--A-TGCTTACAAGCAAGTACAGCAATCAACC-CTCAACTATCACACATCAA-3’
3’-AACTGGTGGACATCATGTATTTTTGGGTTAGGTGTAGTTTT—-G----GGGGA-G-G-GG—-T-ACGAATGTTCGTTCATGTCGTTAGTTGG-GAGTTGATAGTGTGTAGTT-5’
  |225  323|
  |16241 16348|
5’-CTGCAACTCCAAAGCCAC-CCC-TCACCCACTAGGATACCAACAAACCTACCC-ACCCTTAACAGT-ACATAG-TACATAAAGCCATTTACCGTACATAGCACATTACAGTC-3’
3’-GACGTTGAGGTTTCGGTG-GGG-AGTGGGTGATCCTATGGTTGTTTGGATGGG-TGGGAATTGTCA-TGTATC-ATGTATTTCGGTAAATGGCATGTATCGTGTAATGTCAG-5’
  |324  430|
  |16349 <--3’-GTGGTAGGAGGCACTTTAG-5’  16460|
5’-AAATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGGGTCCCTTGACCACCATCCTCCGTGAAATCAATATCCCGCACAAGAGTGCTACTCTCCTCGCTCCGGGCCC-3’
3’-TTTAGGGAAGAGCAGGGGTACCTACTGGGGGGAGTCTATCCCCAGGGAACTGGTGGTAGGAGGCACTTTAGTTATAGGGCGTGTTCTCACGATGAGAGGAGCGAGGCCCGGG-5’
  |431 542|
   |16461  <--3’-GACCAAGGATGAAGTCCCAG-5’ 16569|
5’-ATAACACTTGGGGGTAGCTAAAGTGAACTGTATCCGACATCTGGTTCCTACTTCAGGGTCATAAAGCCTAAATAGCCCACACGTTCCCCTTAAATAAGACATCACGATG-3’-end
3’-TATTGTGAACCCCCATCGATTTCACTTGACATAGGCTGTAGACCAAGGATGAAGTCCCAGTATTTCGGATTTATCGGGTGTGCAAGGGGAATTTATTCTGTAGTGCTAC-5’-end
   |543 651|
 
   16037 – 16368  Highly Variable region in HVSI Conserved sites in HVSI Variable sites in HVSI
 

This figure shows the genomic reference sequence for the extended hyper variable segment I (top line, 5’->3’) and its complement (bottom line, 3’->5’). The mitochondrial DNA (mtDNA) sequence shown here is numbered according to the Revised Cambridge Reference Sequence (rCRS). The numbering of position 1 to 16569, starting at the origin in the control region, refers to the reference strand (top line) in 5’ to 3’ direction, the direction of DNA polymerization during DNA replication. The forward primer’s sequence (‘Left Primer’) is identical with the sequence of the reference strand, and binds therefore on the complement strand (TAACTCCACCATTAGCACCC shown positioned below complement strand). The reverse and sequencing primer’s sequences (‘Right Primer’) are identical to the complement sequence and bind therefore on the reference strand (shown positioned above reference strand). During Polymerase Chain Reaction (PCR) the primers will be extended from the 3’-end (-->). The forward primer will be extended in 5’ to 3’ direction following the direction of the reference strand (in figure to the right) thereby creating a copy of the reference strand. The reverse and sequencing primer will be extended in 5’ to 3’ direction of the complement strand (in figure to the left) creating thereby a copy of the complement strand. The PCR product size is 549 basepairs (bp), which includes the primers themselves and the bp in between the primers (segment 15972-16520). The subsequent sequencing of the PCR product will include bp 15972 to 16400 with a maximum of 429 sequenced bps. Hyper-variable bps are highlighted in yellow, and conserved bps in green. Hyphens (‘-‘) indicate insertions which are hyper-variable.

Table of Primer Statistics: From Program Primer 3

 PCR Primers:   start   end len    tm   gc%  any  3'   sequence 
 LEFT PRIMER 15972  15991  20  57.55 50.00 3.00 0.00   5’-TAACTCCACCATTAGCACCC-3’-->
 Complement      3’-ATTGAGGTGGTAATCGTGGG-5’
 
 RIGHT PRIMER 16520   16501  20  56.25 55.00 3.00 1.00   5’-GACCCTGAAGTAGGAACCAG-3’-->
 Reference     3’-CTGGGCATTCATCCTTGGTC-5’
 
 PCR PRODUCT SIZE: 549, PAIR ANY COMPL:  4.00, PAIR 3' COMPL: 1.00

 Sequencing Primer: start  end len   tm  gc%  any   3'  sequence  
 RIGHT PRIMER 16419  16401  19  58.87 52.63 3.00 0.00   5’-GATTTCACGGAGGATGGTG-3’-->
 Reference      3’-CTAAAGTGCCTCCTACCAC-5’
 

All primers in the above table are shown in 5’ to 3’ direction. This is the convention for referencing, for instance when ordering them. However, in the figure above, the sequences of the reverse and sequencing primers are shown in opposite direction (3’ to 5’ reading from left to right), since they are shown binding to the reference strand and therefore identical with the complement strand. Therefore the sequences of reverse and sequencing primer as written in 5’ to 3’ direction are also known as the reverse complement to distinguish this representation from the complement (in 3’ to 5’ direction) as shown in the figure above.