DNA SEQUENCING

Review: The structure of DNA

Helix Complementary Base Pairing

Review: The structure of DNA

Unzipping

Antiparallel Strands
“The Concept of Base Pairing is Central to All Processes
Involving Nucleic Acids” -B. Lewin

Cytosine
Guanine

From Dr. John C. Perez, Professor and Director of the Natural Toxins Research Initiative, Texas A&M
http://ntri.tamuk.edu/cell/dna.html
 DNA Polymerase Adds Nucleotides
to the 3’ End
• Both DNA & RNA
synthesis occurs 5’ - 3’
• RNA is translated 5’ - 3’
• DNA is sequenced 5’ -
3’
• By convention, DNA
sequence is presented
in the 5’ -3’ direction

http://gened.emc.maricopa.edu/bio/bio181/BIOBK/BioBookDNAMOLGEN.html
 Synthesis of Both Strands of a Specific DNA
Sequence in vitro

5’ 3’
TTGAGAAAGGAATAAGCAGAATTCGTTCCAAAAAGAATGAGCTGTTGTTTGCAGAAATCGAGTATATGC
AACTCTTTCCTTATTCGTCTTAAGCAAGGTTTTTCTTACTCGACAACAAACGTCTTTAGCTCATATACG
3’ 5’
Forward primer dNTPs
5’ 3’
TTGAGAAAGGAATAAGC DNA POL
AACTCTTTCCTTATTCGTCTTAAGCAAGGTTTTTCTTACTCGACAACAAACGTCTTTAGCTCATATACG
3’ 5’

5’ 3’
TTGAGAAAGGAATAAGCAGAATTCGTTCCAAAAAGAATGAGCTGTTGTTTGCAGAAATCGAGTATATGC
AACTCTTTCCTTATTCGTCTTAAGCAAGGTTTTTCTTACTCGACAACAAACGTCTTTAGCTCATATACG
3’ 5’
 Ingredients

• Template DNA
• 5’ primer
• dNTPs (deoxyribonucleoside triphosphate)
• ddATP
• ddTTP ddNTPs
(Dideoxyribonucleoside
• ddCTP
triphosphate)
• ddGTP
• DNA Polymerase
 DNA Sequencing

5'-GAATGTCCTTTCTCTAAGTCCTAAG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATGG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATGGTACTTCTAG3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
 DNA Sequencing

CGTAA
CGTAAC
CGTAACC
CGTAACCC
CGTAACCCT
CGTAACCCTT
CGTAACCCTTG
CGTAACCCTTGG
Molecular Cloning
MCS
Bacterial
plasmid vector

Origin of replication

Multiplicity
Thermal Cycle Sequencing