BIO 325 Lecture Notes - Lecture 6: Genomic Library, Dna Ligase, Alternative Splicing

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Search for splice junction sequences
Primary transcripts can be spliced in different ways in different cell
types
Analyze mRNA sequences for alternative splicing
cDNA libraries reveal how a primary transcript is spliced in
particular cell types
o Look for genes by sequencing the mRNAs
Purify the mRNA by looking for the poly-A tail
Reverse transcribe RNA into cDNA (more stable
and easier to sequence than mRNA)
Ligate cDNAs into vector using DNA ligase
Each recombinant plasmid contains a cDNA
Genomic library represents all the DNA of the genome in every
tissue, while cDNA library represents only a fraction of the
genome that is transcribed in that tissue (exons only)
cDNA sequence reveals the amino acid sequence of the protein,
which provides information about the protein function
Search for conservative sequences when compared with another species’
DNA
“Conserved” means functionally important
Gene products are similar in different species because they evolved
from common ancestors
Species relatedness can be detected by sequence comparisons
o Genome sequence
o Protein-coding sequence
o Similarity (conservation) in DNA sequences is a way to
find genes in genome sequences
Most highly conserved protein-coding exons (CDS)
Least highly conserved intergenic regions (between genes)
o In any DNA sequence, 6 reading frames exist
You don’t know which strand is the template strand and which is the
RNA-like strand
Computer searches all 6 reading frames for ORFs
Long ORF is likely an exon
If DNA sequence is random, how long is an average ORF?
o Frequency of stop codons:
TAA (1/4)3
TAG (1/4)3
TGA (1/4)3
Total: 3/64 ~ 1/21 triplets
o Average ORF is 21 triplets = 21 aa
Bioinformatics tools
What can you do with all the DNA sequences?
o Align/compare sequences
o Predict open reading frames (ORFs)
o Predict exon/intron boundaries
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