MCELLBI 104 Lecture Notes - Lecture 4: Expressed Sequence Tag, Shotgun Sequencing, Genomic Library


Department
Molecular And Cell Biology
Course Code
MCELLBI 104
Professor
Xavier Darzacq, Craig Miller, Roberto Zoncu
Lecture
4

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LECTURE 4: SEQUENCING AND ASSEMBLING GENOMES II: ANNOTATION
KEY CONCEPTS:
Annotation
cDNA
Expressed Sequence Tags (EST)
Conservation
Ortholog
Shotgun Sequencing
1. Isolate genomic DNA
2. Fragment DNA and make genomic library of clones
a. Can precisely make size of insert
3. Sequence paired end reads from every clone
4. Collapse overlapping continuous sequence into contigs
5. Use paired end reads to connect contigs into scaffolds
Telomeres and centromeres are both highly repetitive and would make shotgun
sequencing difficult but can figure out where they are located
Lots of repetitive pieces = have larger genome library pieces to skip over
repetitive sequence
Genome sequence doesn’t tell you much about the function of the genome
How can you start to figure it out?
Genome Annotation: process of attaching biological information to genome sequences
(determining which subset of the genome is transcribed)
Average gene has 6.3 transcripts and 20,000 genes
Vast majority of the genome (80%) participates in at least one biochemical RNA
or chromatin event in at least one cell type
These are not necessarily a required function
Much more of your genome regulates gene expression than codes for proteins
How can you start to figure out what parts of your genome are transcribed?
Converting RNA transcripts to DNA
1. Harvest mRNA (blood, spit, purify mRNA)
2. Reverse transcribe first cDNA strand
3. Reverse transcribe second cDNA strand
cDNA can be cloned and sequenced just like genomic DNA
Put into vector and make clones
cDNA library = usually made from same mRNA source
find more resources at oneclass.com
find more resources at oneclass.com
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