Sequenciamento de DNA

Primer

1 A 2 5’ 3’ 5’ 3’ Sanger’s Method: How Terminated OH PO4 H 5’ P R ddNTP Terminated Normal Linking 3 4 5 6 A PO4 2- H 3’ 5’ 2 Phosphodiester bond 3’ dideoxynucleotide Can not react

How DNA Sequence Is Determined? DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis Polyacrylamide Gel Electrophoresis T A G C ATCGATCGAT 32P ATCGATCGA 32P ATCGATCG 32P ATCGATC 32P ATCGAT 32P ATCGA 32P G ATCG 32P C ATC 32P T AT 32P A A 32P But these bands can’t tell us the identity of the terminal nucleotides If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence

How to Obtain DNA Fragments Maxam-Gilbert's Method: Chemical method Destroy → Cleavage 32P ATCGATCG AT 32P ATCGATCGAT 32P ATCG ATCGAT Destroy → Cleavage Non-radioactive (invisible) Specific Reaction to G Sanger's Method: 32P Biosynthetic method Keep on going ATCGA 32P A,T,C,G A Analogue TAGCTAGCTA ATCG Producing various fragments or TAGCTAGCTA STOP Terminated Template ATCG A TAGCTAGCTA