Biology And Biomedical Sciences BIOL 2960 Lecture Notes - Lecture 37: Small Interfering Rna, Rna Silencing, Reverse Genetics
13 Dec 2016
School
Department
20 April 2016
Lecture 37: Genomics
I. Genomics
A. Applied Genomics
1. The ability to predict gene function from gene sequence is limited, so need
“functional” genomics to determine functionality of products of genes in
cell/organism
2. “Reverse” Genetics and Genomics
a. To determine the functions of genes in a genome
b. To do this: sequence genomics → gene mutation (reverse genetics) → mutant
phenotype observed → can determine effect on process of interest
c. One crucial resource for functional genomics is a comprehensive set of null
mutants
• First available with S. cerevisiae
• Done by disrupting gene function by targeted mutagenesis using homologous
recombination
d. However, this engineering is a tedious process and more modern technologies
exist
• Targeted double strand breaks can be used for mutagenesis in homologous
sequences
3. RNA Silencing
a. First discovered in C. elegans, double stranded RNA silences gene function
b. Small interfering RNA (siRNA) is synthesized according to investigator and is
introduced exogenously
c. siRNA is designed to target mRNA from a single gene
d. Designed to pair perfectly with a sequence in the mRNA, resulting in mRNA
cleavage and little or no translatable mRNA
e. siRNA acts as an on/off switch for doing reverse genetics
f. siRNA can be used for high throughput gene knockout analysis of each of the
genes in a genome
g. Compared to mRNA
• From polII transcript; made endogenously from a miRNA gene
• Processed in nucleus & cytoplasm
• Evolved to imperfectly pair with sequences in 3’UTRs of mRNAs, typically
from multiple genes, resulting in partial translational repression
• A rheostat, not an on/ off switch, to regulate gene activity
4. CRISPR/Cas9 Technology can generate specific double strand breaks
a. It is an easily engineered, sequence-specific nuclease for use in eukaryotic cells,
to specifically create double strand breaks anywhere in the genome
b. Components:
• Guide RNA (sgRNA) targets to specific sequence by forming RNA/DNA
hybrid
• Cas9 protein that binds to sgRNA and cuts both DNA strands at target
sequence
c. It is the most important modern technique for reverse-genetics and mutations
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