Primer Design for PCR Amplification and Sequencing Reaction
Extended Hyper-variable Segment I (15919 - 16569) |15919 | | | | | | | | | | 16027|5'-TGTAAaCCggAgAtGAAAACCtttTtCcAAGGaCAaaTCAGAGAAAAAGtCTTTAACTCCACCATTAGCACCCaAAGCTAAGATtCTAATTTAAACTAtTCTCTGTtCT-3'
3'-ACATTtGGccTcTaCTTTTGGaaaAaGgTTCCtGTttAGTCTCTTTTTCaGAAATTGAGGTGGTAATCGTGGGtTTCGATTCTAaGATTAAATTTGATaAGAGACAaGA-5' |1 | | | | |5'-TAACTCCACCATTAGCACCC-3'-->| | | 109|
|16028 | | | | | | | | | | 16136|
5'-TTCaTGGGGaagCagATTTGgGtacCACCCAagtAtTGacTcaccCatcAAcaaccgctATgTAtttcGtaCATtActgccagcCaccAtgaaTattGtaCagtaccat-3'
3'-AAGtACCCCttcGtcTAAACcCatgGTGGGTtcaTaACtgAgtggGtagTTgttggcgaTAcATaaagCatGTAaTgacggtcgGtggTacttAtaaCatGtcatggta-5' |110 | | | | | | | | | | 218| |16137 | | | | | | | | | | 16245|5'-aaatAcTtgaccAcCtgtagtaCataaaaacccaatccacatcaaaaccctccccccatgctTAcAagcaagtacagcaatcaaccttcAactgtcacacaTcaaCtgc-3'
3'-tttaTgAactggTgGacatcatGtatttttgggttaggtgtagttttgggaggggggtacgaATgTtcgttcatgtcgttagttggaagTtgacagtgtgtAgttGacg-5' |219 | | | | | | | | | | 327| |16246 | | | | | | | | | | 16354|5'-aactcCaaagccacccctcacCcactaggaTatcaaCAaacctacccacccttaacagtacAtagcacaTaaagccatttaccGtacatagcacaTtacagtcaaatcc-3'
3'-ttgagGtttcggtggggagtgGgtgatcctAtagttGTttggatgggtgggaattgtcatgTatcgtgtAtttcggtaaatggCatgtatcgtgtAatgtcagtttagg-5'
|328 | | | | | | | | | | 436| |16355 | | | | | | | | | | 16463|5'-cttctcgtccccatGGaTGAcCcCCctCAgATAggggTCCcTTgacCACCATCCTCCGTGAAAtcAAtAtCCCgcACAAGAGTgCtACTCTCCTCGCTCCGgGCCCATa-3'
3'-gaagagcaggggtaCCtACTgGgGGgaGTcTATccccAGGgAActgGTGGTAGGAGGCACTTTAgTTaTaGGGcgTGTTCTCAcGaTGAGAGGAGCGAGGCcCGGGTAt-5' |437 | | | | | | | | | | 545| |16464 | | | <--3'-GACCAAGGATGAAGTCCCGG-5' | | | |16569|5'-AcACtTggGgGTAGCTAAagTGAAcTGTATCCGaCAtCTGGTTCCTACTTCAGGgcCATAaAgcCTAAATAGCCCAcACGTTCCcCTTAAATAAGACATCACGaTG-3'-end
3'-TgTGaAccCcCATCGATTtcACTTgACATAGGCtGTaGACCAAGGATGAAGTCCcgGTATtTcgGATTTATCGGGTgTGCAAGGgGAATTTATTCTGTAGTGCtAC-5'-end |546 | | | | | | | | | | 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 Reconstructed Sapiens Reference Sequence (RSRS 2012 Paper) (Full Sequence) (HVSII Sequence). 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 16500 with a maximum of 529 sequenced bps.
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'-GGCCCTGAAGTAGGAACCAG-3'-->
Reference 3'-CCGGGCATTCATCCTTGGTC-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 16520 16501 20 56.25 55.00 3.00 1.00 5'-GGCCCTGAAGTAGGAACCAG-3'-->
Reference 3'-CCGGGCATTCATCCTTGGTC-5'
MAX SEQUENCING SIZE: 529 (15972-16500)
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.