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1
Basic Biology Related to Technology for Measuring
Gene Expression
1/11/2011
Copyright © 2011 Dan Nettleton
2
Central Dogma
DNA
RNA
protein
(transcription)
(translation)
Proteins perform essential biological functions.
DNA contains genes that code for proteins.
3
A VERY Simplified Eukaryotic Cell
nucleus
chromosome
DNA strands
DNA contains thousands of genes.
cytoplasm
4
Deoxyribonucleic Acid (DNA)P
S
P
S
P
S
P
S
P
S
P
S
P
S
P
S S S S S S S
P P P P P P P P
5’ 3’
5’3’
A A T T G C C
T T A A C G G
S=deoxyribose sugarP=phosphate
BasesA=adenineC=cytosineG=guanineT=thymine
hydrogenbonds
The two DNA strands are twisted inthe famous double helix shape (basepairs like rungs in a spiraling ladder).
......
... ...
purinespyrimidines
5
Nucleotide=Phosphate-Sugar-BaseP
S
P
S
P
S
P
S
P
S
P
S
P
S
P
S S S S S S S
P P P P P P P P
5’ 3’
5’3’
A A T T G C C
T T A A C G G
......
... ...
6
Sequence of bases holds instructions for manufacturing proteins
P
S
P
S
P
S
P
S
P
S
P
S
P
S
P
S S S S S S S
P P P P P P P P
5’ 3’
5’3’
A A T T G C C
T T A A C G G
......
... ...
CodingSequence
2
7
RNA Polymerase
• An enzyme is a protein that catalyzes chemical reactions.
• RNA polymerase is the enzyme responsible for transcribing DNA to RNA.
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Transcription
P
S
T
P
S
A
P
S
A
P
S
C
P
S
G
P
S
P
3’
G
...P
S5’
T
...
S S S S S S S
P P P P P P P P
5’3’
A A T T G C C
... ...
9
Transcription
P
S
A
P
S
A
P
S
C
P
S
G
P
S
U
P
S
P
3’
G
...P
S5’
U
...
S
P
A
S
P
T
S
P
T
S
P
G
S
P
C
S
P
3’
A
... S
P P
5’
C
...
10
Ribonucleic Acid (RNA)
P
S
A
P
S
A
P
S
C
P
S
G
P
S
U
P
S
P
3’
G
...P
S5’
U
...
BasesA=adenineC=cytosineG=guanineU=uracil
S=ribose sugarP=phosphate
The product of transcriptionis known as the
primary transcript.
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Posttranscriptional Modificationsto Primary Transcript
Primary transcript
Intervening sequences corresponding to intronsthat are removed through splicing
3’ UTR5’ UTR
Primary transcript after modification: messenger RNA (mRNA)
AAAAAA...AAAA
poly-A tailCoding portions of RNA sequencecorresponding to exons
5’ UTR 3’ UTR
5’ cap
G
12
Expression Levels
• The amount of messenger RNA (mRNA) produced by a gene is known as the gene's expression level.
• This course focuses on the design and analysis of experiments that involve the simultaneous measurement of gene expression levels for thousands of genes.
3
13
Transcription takes place inside the nucleus.
nucleus
chromosome
DNA strands cytoplasm
Translation takes place outside the nucleus.14
Translation
mRNA
Ribosome
amino acid sequence
folds to become a protein
15
During translation transfer RNA (tRNA) translates the genetic code
... ...A A C GU GU
codon codon
A A U
leu
U G C
thr
tRNAanticodon
amino acids
16
The Genetic Code
UUU phe UCU ser UAU tyr UGU cysUUC phe UCC ser UAC tyr UGC cysUUA leu UCA ser UAA STOP UGA STOPUUG leu UCG ser UAG STOP UGG trp
CUU leu CCU pro CAU his CGU argCUC leu CCC pro CAC his CGC argCUA leu CCA pro CAA gln CGA argCUG leu CCG pro CAG gln CGG arg
AUU ile ACU thr AAU asn AGU serAUC ile ACC thr AAC asn AGC serAUA ile ACA thr AAA lys AGA argAUG met ACG thr AAG lys AGG arg
GUU val GCU ala GAU asp GGU glyGUC val GCC ala GAC asp GGC glyGUA val GCA ala GAA glu GGA glyGUG val GCG ala GAG glu GGG gly
Firs
t Bas
eSecond Base
U
C
A
G
U C A G
mRNAcodon
aminoacid
17
Miscellaneous Comments
• The biology is more complicated than I described.
• It is amazing!
• Humans have somewhere around 30,000 genes. (The exact number is a subject for debate.)
• Much of the variation is created by differences in how cells use the genes they have.
• Microarrays and Next Generation Sequencing are tools that can help us understand how cells of various types use their genes in response to varying conditions. This helps us to understand gene function.