Sequencing DNA – the Sanger Method – the most-popular way (one of many methods) A primer binds to one of the DNA strands at a specific location (such as the Cyto B gene) Nucleotides (dNTPs) are floating around in solution A DNA polymerase begins to synthesize a new stand of DNA Then a “fluorescently-tagged” ddNTP (di-deoxynucleotide triphosphate) gets incorporated into the growing chain of DNA but terminates the chain (it cannot be elongated). Scientists use 4 different color “glowing” ddNTPs: One color for each of the 4 bases of DNA (eg. green-glowing “A”, blue glowing “C”, etc.). The DNA fragments – all copied to different lengths – are analyzed by “capillary” gel electrophoresis. Smaller fragments run faster. Fragments arrange by size! A detector “reads” the color of light as each DNA fragment flows past.

Cytochrome B sequencing (1 person)

Text format of DNA sequencing Comparison of Cyt B between samples 01 and 02: (1 difference in 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACCATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 Text format of DNA sequencing A comparison between 3 individual’s Cytochrome B gene (303 bases) 1 base difference between persons 1 and 2 Comparison of Cyt B between samples 01 and 03: (2 differences in 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACCATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 2 base differences between persons 1 and 3 Comparison of Cyt B between samples 02 and 03: (1 difference in 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 1 base difference between persons 2 and 3

A comparison between 3 humans and Neanderthal Comparison of Cyt B between sample 01 and Neanderthal: (3 differences out of 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCCGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACCATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 A comparison between 3 humans and Neanderthal in the Cytochrome B gene (303 bases) 3 base differences between person 1 and Neanderthal Comparison of Cyt B between sample 02 and Neanderthal: (2 differences out of 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCCGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGGCGAGGCCTATAT 240 ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 2 base differences between person 2 and Neanderthal Comparison of Cyt B between sample 03 and Neanderthal: (1 difference out of 303 bases) Query 1 CCATCCAACATCTCAGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 |||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CCATCCAACATCTCCGCATGATGAAACTTCGGCTCACTCCTTGGCGCCTGCCTGATCCTC 60 Query 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 61 CAAATCACCACAGGACTATTCCTAGCCATGCACTACTCACCAGACGCCTCAACCGCCTTT 120 Query 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Sbjct 121 TCATCAATCGCCCACATCACTCGAGACGTAAATTATGGCTGAATCATCCGCTACCTTCAC 180 Query 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Sbjct 181 GCCAATGGCGCCTCAATATTCTTTATCTGCCTCTTCCTACACATCGGACGAGGCCTATAT 240 Query 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 241 TACGGATCATTTCTCTACTCAGAAACCTGAAACATCGGCATTATCCTCCTGCTTGCAACTATA 303 1 base difference between person 3 and Neanderthal

Video about sequencing and the Human Genome Project: PBS.org>Gene Reader Secrets of the sequence - Missing Gene in Breast Cancer video Timothy Ray Brown – Berlin Patient – Video