Next Generation Sequencing

Sanger Dideoxy Method (1st Generational)

Basic Structure of Nucleotides

• A nucleotide is a polymer of DNA

– Phosphate

– Sugar

• Deoxy sugar

– Nucleoside

• A, T, G, C

http://myhome.sunyocc.edu/~weiskirl/nucleotides_nucleicacid.htm

DNA Synthesis

• Phosphate (5ˈ) OH (3ˈ)

http://myhome.sunyocc.edu/~weiskirl/nucleotides_nucleicacid.htm

Dideoxynucleotides

• Di-deoxy 2ˈ and 3ˈ are deoxy (without OH)

– Di 2

Using the Dideoxynucleotides

• DNA sample divided into four separate

sequencing reactions• Each reaction has only one of the four

dideoxynucleotides

http://www.jenabioscience.com/cms/en/1/catalog/605_dideoxynucleotides_ddntps.html

Gel Electrophoresis

• Denaturation (95° C)

• Gel Electrophoresis• Size separation for length

• Dye Fluorescence• Detected by sequencing

machine as fluorescent

trace chromatograms

Work Cited

• Ambry Genetics. Making Sense of NextGent Sequencing. Kelly Gonzalez, MS,

CGC, and Senior Manager of Clinical Genomics. Available at

http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10-

12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May

21, 2014.

• Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project.

Next Generation Sequencing. Available at http://www.omixon.com/the-basics-

of-next-generation-sequencing/. Access verified May 21, 2014.

• Chapter 2 Notes: Organic Chemistry.

http://mayhewbiology.com/Biology%20notes/carbo%20notes.htm Access

verified July 30, 2014.

• OCC Biology. Nucleic acids contain genetic information and enable synthesis of

proteins. Nucleotides are used to make Nucleic Acids.

http://myhome.sunyocc.edu/~weiskirl/nucleotides_nucleicacid.htm Access

verified July 30, 2014.

• Sanger sequencing. (2014, July 27). In Wikipedia, The Free Encyclopedia.

Retrieved 19:34, July 30, 2014, from

http://en.wikipedia.org/w/index.php?title=Sanger_sequencing&oldid=618681657

Access verified July 30, 2014.

Recommended Videos

Sanger method of DNA sequencing, 3D

animation with narration

http://www.dnalc.org/view/15479-Sanger-

method-of-DNA-sequencing-3D-

animation-with-narration.html

Dideoxy DNA Sequencing

https://www.youtube.com/watch?v=bEFL

Bf5WEtc

Next Generation Sequencing

454-Pyrosequencing