Biology 1002B Study Guide - Final Guide: Pyrosequencing, Dna Ligase, Hybridization Probe
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DNA Sequencing- determining the precise order of DNA sequence with its nucleotides
Common Steps of DNA sequencing
1. Purification
2. Fragmentation of DNA
3. Amplification
4. Sequencing Fragments
5. Assembling into genomic sequence
1. Sanger Dideoxy method for DNA sequencing
DNA strand obtained
Primer added
Nucleotide mixture added- 99% deoxyribonucleotide and *1 %
dideoxyribonucleotide
4 tubes prepared each with 1% ddATP, ddGTP, ddCTP, ddTTP
Dideoxynucleotide have no hydroxyl group at 3’ end
Once dideoxy is attached, no elongation of complementary strand
Result: different sized fragments in one tube- sizes show where the T or A or G or C
is added
Shortest sequence/ fragment- first base
4 tubes undergo electrogelphoresis to know exact sequencing
2. Modern Automated Sanger
Instead of 4 tubes, only one is used
Dideoxynucleotides are tagged with florescent
Color band shows sequencing and nucleotide order
3. Pyrosequencing
Pyrophosphate is released when nucleotide is attached to the growing strand
Enzyme is present to convert pyrophosphate into ATP
ATP is used to make light under enzymes
Flash of light represents the addition of another base
Adapter added to each strand of double strand DNA- allows for adhesion on to bead
surface
Strands attached to beads undergoes PCR
Each beads with thousands of same DNA copy goes into wells
Each bead contain different fragments
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