gene discovery
TRANSCRIPT
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7/24/2019 Gene Discovery
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Application ofrecombinant DNAtechnology and
forward/reversegenetics
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7/24/2019 Gene Discovery
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Applied molecular biology
Treatment of diabetes with synthetic insulin
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7/24/2019 Gene Discovery
3/23
Clone the insulin gene for expression inbacteria
insulin cDNA
oriV
ampR
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7/24/2019 Gene Discovery
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cDNA vs genomicDNA
cDNA contains the sequences in processedmRNAExons ntrons
!romoter
"# $TR
DNA
!re%mRNA
mRNA AAAAAAAAAAA
$TR 'untranslated region(
cDNA TTTTTTTTTTT
Exons
Transcription
Reversetranscripti
on
Processing
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7/24/2019 Gene Discovery
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Making cDNA
solation of mRNA from other cellular RNAs
TTTTTTT
TTTTTTT
TTTTTTT
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAA
AAAAAA
AAAAAAA
AAAAAAA
AAAAAA
AAAAAAA
)ligo%dT
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7/24/2019 Gene Discovery
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Making cDNA
*ynthesising +rst strand of cDNA from RNA Re,erse transcriptase- an RNA%dependent DNA
polymerase
AAAAAAAAA
TTTTTTTTT
"#
)ligo%dT
primerAAAAAAAAA
TTTTTTTTT
"#
RT
TTTTTTTTTAAAAAAAAA
"#
"
#
Retro,irus
RT
.)%
Treat
withRNase
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7/24/2019 Gene Discovery
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Amplify insulin cDNA byPCR
"#
"#
TTTTTTTTT
dAT!/ d0T!/ dCT!/ dTT!Taq polymerase
TTTTTTTTT
TTTTTTTTT
TTTTTTTTT
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7/24/2019 Gene Discovery
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igate into vector and transform bacteria
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7/24/2019 Gene Discovery
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!ene discovery
0ene sequence0ene function
DNA sequence
!henotype
.omologous gene
Dont haveany sequenceinformation
Have somesequenceinformation
Have thegenessequence
Understand thegenes function
0enomesequenc
e!rotei
n
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7/24/2019 Gene Discovery
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!ene discovery " forwardgenetics
0ene sequence0ene function
DNAsequence
!henotype
.omologous
gene
Dont haveany sequenceinformation
Have somesequenceinformation
Have thegenessequence
Understand thegenes function
0enomesequenc
e!rotei
n
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7/24/2019 Gene Discovery
11/23
Review# terminology ocus'pl1 loci( 2
location of a geneor other DNAsequence in a
genome !ene2 a
functional DNAsequence in a
genome Allele2 a ,ariant
of the DNA
sequence at ai,en locus
A locusThere is a gene here
Another locusThere is another gene here
And another locus
No gene exists here
An allele Another allele
An allele *ame allele
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7/24/2019 Gene Discovery
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Molecular markers
Markersare loci that act as indicators orsignposts Molecular markers are basically a speci+c DNA
sequence at a 3nown location on a chromosome Restriction fragment length polymorphisms 'R45!s( are
simple molecular mar3ers
CT
GC
AG
GA
CG
TC
CT
GTA
G
GA
CAT
C
CT
GC
AG
GA
CG
TC
CT
GC
AG
GA
CG
TC
CT
GC
AG
GA
CG
TC
CT
GC
AG
GA
CG
TC
!olymorphism
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7/24/2019 Gene Discovery
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$orward genetics 4rom phenotype to gene 2 6classical7
genetics *tudy phenotypes of 6mutants7 to wor3 out
genetic basis8ild%type )bese
9ice- http-::www1hhmi1org:genesweshare:d;&
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7/24/2019 Gene Discovery
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$orward genetics >reeding experiments to +nd and con+rm
a molecular mar3er that is ?lin3ed# to thephenotype-The DNA sequence around this molecular
mar3er can then be determined to +nd the Obgene sequence
5in3age mapping
GC
TA
GC
TA
GT
CG
TA
GC
TA
GG
AT
GC
TA
CCG
ATAT
CGCG
ATA
GC
TC
TA
GG
AOb locus
D=Rc3;& locus
Determine DNA
sequence
9ice- http-::www1hhmi1org:genesweshare:d;&
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7/24/2019 Gene Discovery
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!ene discovery " forwardgenetics
0ene sequence0ene function
DNAsequence
!henotype
.omologousgene
Dont haveany sequenceinformation
Have somesequence
information
Have thegenes
sequence
Understand thegenes function
0enomesequenc
e!rotei
n
di
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7/24/2019 Gene Discovery
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!ene discovery " reversegenetics
0ene sequence0ene function
!rotein
DNAsequence
!henotype
.omologousgene
Dont haveany sequenceinformation
Have somesequence
information
Have thegenes
sequence
Understand thegenes function
0enomesequenc
e
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7/24/2019 Gene Discovery
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Reverse genetics
CGATATGGCGATACGAT
GCTACAATGCGACCACG
TAGCTATGCAGTCGTAG
TAGTAGCGGTAGCCCCC
AGTACCGGAGGGGCGGT
AGCTAGCTAGCCCCGGC
TAGTCGAGGCTAGCTGA
TCATCTGGCGGATTAGC
GGATCGATACGGCCGCGCACTATCATGCTAGCTC
GCGCGGAGCTAGCTAGT
ACGCATGACTGCATGCA
TGCATGCATGACTGCAT
9utate
*equencegenome
bidopsis images- D) ;
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7/24/2019 Gene Discovery
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Reverse genetics "%nding a gene
*equence the genome/ typically ofmodel organisms 9any sequencing techniques
>ene+ts of using model organisms
Easy to manipulate genetically Easy to grow/ short life cycles
8hy can#t we Bust stop at thegenome sequence A long string of A/ T/ 0/ C doesn#t
meananything
Need to +nd genes within the letters/and disco,er their function
us musculus
Drosophila
east
!" coli
#rabidopsis
R i
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7/24/2019 Gene Discovery
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Reverse genetics "%nding a gene
*earch the genomedata 4ind homologous
genes *earch genome using
the sequence ofanother gene:proteinof similar function
!redict new genes
by loo3ing for- *tart and stop codons
Transcription translation initiation
ATG (NNN)nTAA
TAG
TGA
0ene sequence
!rotein
.omologousgene
Have somesequence
information
Have thegenes
sequence
0enomesequenc
e
CCAATTATA
Kozak consensus sequence
Shine-Dalgarno sequence
Polyadenylaion signals
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7/24/2019 Gene Discovery
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Reverse genetics " from geneto function
Disco,er the gene#s function by editing it E1g1 ma3e a transcriptional fusion
!romoter for gene of interest is cloned next to a?reporter# gene
E1g1 ma3e a translational fusion 0ene of interest is
cloned next to the04! gene
C!F;