Application of

recombinant DNA
technology and
forward/reverse
genetics

Applied molecular biology

Treatment of diabetes with synthetic insulin

 Clone the insulin gene for expression in

bacteria

insulin cDNA

ampR

oriV

cDNA vs genomic
DNA

cDNA contains the sequences in processed

mRNA
Exons
Introns
DNA
Transcription

Promoter

Pre-mRNA
Processing
mRNA
Reverse
transcripti
on
cDNA

AAAAAAAAAAA

5 UTR

3 UTR (untranslated region)

TTTTTTTTTTT

Exons

Making cDNA
Isolation of mRNA from other cellular RNAs
AA

AA

AA

AA

Oligo-dT

AAAAAAAAA

AA

AA

A
AA

TTTTTTT
AAAAAAA

TTTTTTT
AAAAAA

AA A A A A A

AA AA

AA

AAAAAAA

TTTTTTT
AAAAAAA
AA

AA

AA

AA

Making cDNA
Synthesising first strand of cDNA from RNA
Reverse transcriptase: an RNA-dependent DNA
polymerase
Retrovirus

RT

OligodT
primer

AAAAAAAAA
TTTTTTTTT

RT
3

AAAAAAAAA
TTTTTTTTT

5
3 HO-

AAAAAAAAA
TTTTTTTTT

Treat
with
RNase

Amplify insulin cDNA by

PCR
3

TTTTTTTTT

TTTTTTTTT

Taq polymerase

dATP, dGTP, dCTP, dTTP

TTTTTTTTT

TTTTTTTTT

Ligate into vector and transform bacteria

Gene discovery
Dont have
any sequence
information

Have some
sequence
information

Have the
genes
sequence

Understand the
genes function

Homologous gene

Phenotype

Gene sequence

DNA sequence

Protei
n

Genome
sequenc
e

Gene function

Gene discovery forward

genetics
Dont have
any sequence
information

Have some
sequence
information

Have the
genes
sequence

Understand the
genes function

Homologous
gene
Phenotype

Gene sequence

DNA
sequence
Protei
n

Genome
sequenc
e

Gene function

Review: terminology
Locus (pl. loci)
An allele
location of a gene
A locus
or other DNA
There is a gene here
sequence in a
Another locus
genome
There is another gene here
Gene a
functional DNA
sequence in a
And another locus
No gene exists here
genome
Allele a variant
of the DNA
An allele
sequence at a
given locus

Same allele

Another allele

Molecular markers
Markers are loci that act as indicators or
signposts
Molecular markers are basically a specific DNA
sequence at a known location on a chromosome
CTGCAG
GACGTC

CTGCAG
GACGTC

CTGTAG
GACATC

CTGCAG
GACGTC

CTGCAG
GACGTC

CTGCAG
GACGTC

Restriction fragment length polymorphisms (RFLPs) are

simple molecular markers

Polymorphism

Forward genetics
From phenotype to gene classical
genetics
Study
phenotypes
Wild-type
Obese of mutants to work out
genetic basis
brown

Mice: http://www.hhmi.org/genesweshare/d130.html

D6Rck13

Identify molecular
markers that are
inherited together with
the phenotype being
investigated

 Breeding experiments to find and confirm

a molecular marker that is linked to the
phenotype:
The DNA sequence around this molecular
marker can then be determined to find the Ob
gene sequence
Linkage mapping
Ob locus
D6Rck13 locus

Phenotype
Mice: http://www.hhmi.org/genesweshare/d130.html

Determin
e DNA
sequence

GCTAGCTAGTCGTAGCTAGGATGCTACCGATATCGCGATAGCTCTAGGA

Forward genetics

Gene sequence

Gene discovery forward

genetics
Dont have
any sequence
information

Have some
sequence
information

Have the
genes
sequence

Understand the
genes function

Homologous
gene
Phenotype

Gene sequence

DNA
sequence
Protei
n

Genome
sequenc
e

Gene function

Gene discovery reverse

genetics
Dont have
any sequence
information

Have some
sequence
information

Have the
genes
sequence

Understand the
genes function

Homologous
gene
Phenotype

Gene sequence

DNA
sequence
Protei
n

Genome
sequenc
e

Gene function

Reverse genetics
From gene to phenotype in the functional
genomics era
Sequence a genome and find a gene that
interests you
Then, study theCGATATGGCGATACGAT
function of the gene by changing
GCTACAATGCGACCACG
the gene (expression,
sequence, etc) and seeing
TAGCTATGCAGTCGTAG
TAGTAGCGGTAGCCCCC
what happens
Sequence AGTACCGGAGGGGCGGT

abidopsis images: DOI 10.1104/pp.110.157545

genome

AGCTAGCTAGCCCCGGC
TAGTCGAGGCTAGCTGA
TCATCTGGCGGATTAGC
GGATCGATACGGCCGCG
CACTATCATGCTAGCTC
GCGCGGAGCTAGCTAGT
ACGCATGACTGCATGCA
TGCATGCATGACTGCAT

Genome
sequenc
e

Mutate

Gene
sequence

Gene
function

Reverse genetics
finding a gene

Sequence the genome, typically of

model organisms

Mus musculus

Many sequencing techniques

Benefits of using model organisms

Easy to manipulate genetically
Easy to grow, short life cycles

Why cant we just stop at the

genome sequence?
A long string of A, T, G, C doesnt
mean anything
Need to find genes within the letters,
and discover their function

Arabidopsis

Drosophila

Yeast

E. coli

Reverse genetics
finding a gene

Search the genome

data
Find homologous
genes

Search genome using

the sequence of
another gene/protein
of similar function

Predict new genes

ATG (NNN) for:
TAA
by looking

Have some
sequence
information

Have the
genes
sequence

Homologous
gene

Gene sequence

Protei
n

Genome
sequenc
e

Start and

TAG
stop
TGA

codons

Transcription &
translation initiation

CCAATTATA
Kozak consensus sequence
Shine-Dalgarno sequence
Polyadenylation signals

Reverse genetics from gene

to function
Discover the genes function by editing it
E.g. make a transcriptional fusion
Promoter for gene of interest is cloned next to a
reporter gene

E.g. make a translational

CPK10
GFP
fusion
Gene of interest is
cloned next to the
GFP gene

PK10 paper: DOI 10.1104/pp.110.157545

Reverse genetics from gene

to function
Discover the genes function by making
mutants
CPK10

Wild-type

CPK10

Knockout
mutant
under
drought
stress

CPK10

Overexpressi
on mutant
under drought
stress

Gene sequence

Wild-type
under
drought

PK10 paper: DOI 10.1104/pp.110.157545

Gene function

Gene discovery reverse

genetics
Dont have
any sequence
information

Have some
sequence
information

Have the
genes
sequence

Understand the
genes function

Homologous
gene
Phenotype

Gene sequence

DNA
sequence
Protei
n

Genome
sequenc
e

Gene function

Learning objectives
Differentiate between the content and application
of genomic DNA and cDNA
Differentiate between forward and reverse
genetics
Describe the principles that lead to gene
identification, and the processes that allow
discovery of gene function