LIFESCI 23L Lecture Notes - Lecture 10: Clustal, Phylogenetic Tree, Sanger Sequencing
Lecture J: DNA Sequencing
Skills/Concepts
● Receive mitochondrial DNA sequence
● Overview:
○ Clean up raw data in Trace Editor
○ estimate Haplogroup using ClustalW
○ use Mito-Blast to validate haplogroup
○ Annotate mtDNA sequence
○ Align sequence with other similar sequences
○ Create a phylogenetic tree with your sequence
■ With selected mtDNA sequences from database
■ With sequences from class
○ Troubleshooting and class discussion or results
● Skills
○ Using Mega5, BLAST, annotating DNA, using ClustalW, drawing a phylogenetic tree
● Concepts:
○ Unerstanding DNA sequencing methods and human migration patterns and interpreting
phylogenetic trees
Background Information
● Lecture Overview
● Human Origin
○ We carry about 1-4% of neaderthal DNA
● Human Migration
● DNA Sequencing
○ Haplotype = specific nucleotide sequence of mitochondrial genome
○ Haplogrou = all indviduals with the same haplotype along with similar haplotypes
○ Sanger Sequecing
■ Requires template DNA (with DNA to be sequenced, typically amplified)
■ Requires polymerase (does not have to be heat-stable)
■ Requires sequencing primer → extended by polymeras; can be radioactively probes
■ Requires nucleotides; not only nucleotides but dideoxynucleotides (in low concentration)
● Causes STOP in sequencing at various places → produces many strands of
different lengths which can be read in order using different size
● Each ddnucleotide could also be labelled with fluorescent tags → able to be
sequenced by color (by a computer! → automated)
● DNA sequence analysis
○ Sanger method sufficient for routine sequencing
○ More ambitious sequencing (e.g. genome sequencing) → other techniques
■ Capillary sequences, pyrosequencing,
● Example Annotation
● Further Explorations
Lab Details
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Clean up raw data in trace editor. Create a phylogenetic tree with your sequence. Using mega5, blast, annotating dna, using clustalw, drawing a phylogenetic tree. Unerstanding dna sequencing methods and human migration patterns and interpreting. We carry about 1-4% of neaderthal dna. Haplotype = specific nucleotide sequence of mitochondrial genome. Haplogrou = all indviduals with the same haplotype along with similar haplotypes. Requires template dna (with dna to be sequenced, typically amplified) Requires polymerase (does not have to be heat-stable) Requires sequencing primer extended by polymeras; can be radioactively probes. Requires nucleotides; not only nucleotides but dideoxynucleotides (in low concentration) Causes stop in sequencing at various places produces many strands of different lengths which can be read in order using different size. Each ddnucleotide could also be labelled with fluorescent tags able to be sequenced by color (by a computer! More ambitious sequencing (e. g. genome sequencing) other techniques.
