Transcript Figure 20.2 Overview of gene cloning with a bacterial

Figure 20.2 Overview of gene cloning with a bacterial
plasmid, showing various uses of cloned genes
Bacterium
1 Gene inserted
Cell containing gene
of interest
into plasmid
Bacterial
chromosome
Gene of
interest
Plasmid
Recombinant
DNA (plasmid)
2 Plasmid put into
DNA of
chromosome
bacterial cell
Recombinate
bacterium
3 Host cell grown in culture,
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein expressed
by gene of interest
Protein harvested
4 Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth
hormone treats
stunted growth
Figure 20.3 Using a restriction enzyme and DNA ligase to
make recombinant DNA
Restriction site
DNA 5
3
3
5
GAATTC
CTTAAG
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow
G
G
Sticky end
2 DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
G AATTC
C TTAA G
G
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATTC
CTTAA G
One possible combination
3 DNA ligase
seals the strands.
Recombinant DNA molecule
Figure 12.3 Cloning a gene in a bacterial plasmid
Human cell
E.coli
1 Isolate DNA
from two sources
2 Cut both DNAs
with the same
restriction enzyme
DNA
Plasmid
Gene V
Sticky ends
3 Mix the DNAs;
they join by
base-pairing
4 Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
5 Put plasmid into bacterium
by transformation
Recombinant
bacterium
6 Clone the bacterium
Bacterial clone carrying many
copies of the human gene
Genomic libraries
Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA
or
Bacterial
clone
Plasmid library
Phage
clone
Phage library
Making an intron-lacking gene from
eukaryotic mRNA
Cell nucleus
Exon Intron
Exon
Intron Exon
DNA of
eukaryotic
gene
1 Transcription
RNA
transcript
2 RNA splicing
(removes introns)
mRNA
Test tube
Reverse transcriptase
3 Isolation of mRNA
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
cDNA strand
4 Breakdown of RNA
5 Synthesis of second
DNA strand
cDNA of gene
(no introns)
Table 12.6 Some protein products of
recombinant DNA technology
A DNA probe tags a gene by base
pairing
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Base pairing
indicates the
gene of interest
PCR
5
3
Target
sequence
3
Genomic DNA
1 Denaturation:
5
5
3
3
5
Heat briefly
to separate
DNA strands
2 Annealing:
Cycle 1
yields
2
molecules
Cool to allow
primers to
hydrogen-bond.
Primers
3 Extension:
DNA polymerase
adds nucleotides
to the 3 end of
each primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
New
nucleotides
PCR
Primitive PCR machine
Applications for PCR
•
•
•
•
DNA cloning for sequencing
Functional analysis of genes
Diagnosis of genetic diseases
ID genetic fingerprints (i.e. forensics and
paternity testing)
• Detection and diagnosis of infectious
diseases (e.g. H1N1)
Gel electrophoresis of DNA
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Completed gel
Lane 1 – Father
Lane 2 – Child
Lane 3 – Mother
The child has
inherited some,
but not all of the
fingerprint of each
of its parents,
giving it a new
and unique
fingerprint.
Gel box and power source
Gel Electrophoresis Plate
DNA fingerprints from a murder case
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4 g
D
Jeans
shirt
Victim’s
blood (V)
8 g
V
“Pharm” animals