Download Chapter 17 Recombinant DNA and Biotechnology

Recombinant DNA
&
Biotechnology
Recombinant DNA
• recombinant DNA molecules contain DNA
from different organisms
– any two DNAs are joined by DNA ligase
5’GGATCATGTA-OH
3’CCTAGTACAT-P
P-CCCGATTTCAAT
HO-GGGCTAAAGTTA
DNA ligase
5’GGATCATGTACCCGATTTCAAT
3’CCTAGTACATGGGCTAAAGTTA
figure 17-01.jpg
restriction enzymes degrade
invading viral DNA
Figure 16.1
Cleaving and Rejoining DNA
• RE produce many different DNA fragments
restriction enzymes recognize specific
DNA sequences (recognition sites)
EcoRI
5’GGATCGAATTCCCGATTTCAAT
3’CCTAGCTTAAGGGCTAAAGTTA
a palindrome reads the same
left-to-right in the top strand
and
right-to-left in the bottom strand
staggered cuts produce “sticky ends”
Figure 16.4
Cutting and Rejoining DNA
• restriction enzymes (RE) produce specific
DNA fragments for ligation
– RE are defensive weapons against viruses
– RE “cut” (hydrolyze) DNA at specific sites
– RE “staggered cuts” produce “sticky ends”
– sticky ends make ligation more efficient
gel
electrophoresis
Figure 16.2
Cleaving and Rejoining DNA
• RE produce many different DNA fragments
– for a 6 bp recognition site
1/46 = 1/4096 x 3x109 bp/genome =
7.3 x105 different DNA fragments
• gel electrophoresis sorts DNA fragments by
size
• hybridization with a labeled probe locates
specific DNA fragments
Southern
hybridization
of a
labeled
probe
to a
DNA
target
Figure 16.3
gel electrophoresis & Southern hybridization
Cloning Genes
• genetic engineering requires lots of DNA
– cloning produces lots of exact copies
– DNA clones are replicated by host cells
– DNA is cloned in a DNA vector
– a DNA vector has an origin of replication
(ori) that the host cell recognizes
pBR322 is a historical bacterial cloning plasmid
a Yeast Artificial Chromosome vector has yeast
ori, centromere and telomeres
Agrobacterium Ti plasmid has an
Agrobacterium
ori and T DNA that integrates into plant DNA
Figure 16.5
Cloning Genes
• a DNA vector with its ligated insert must be
introduced into the host cell
• chemical treatment makes cells “competent”
- ready for heat shock transformation
• electroporation opens pores in the plasma
membrane
• mechanical treatment inserts DNA
physically
Cloning Genes
• vectors carry reporter genes
– antibiotic resistance protects host cells that
carry a vector (selection)
– proteins such as -galactosidase, luciferase
or Green Fluorescent Protein (GFP) identify
transformed cells (screening)
bacterial plasmid pBR322
is a
cloning vector
that encodes
ampicillin & tetracycline
antibiotic resistances
insertion of a target DNA inactivates tetracycline
resistance
Figure 16.6
ligating vector to insert
~4300 bp; 0.1 µg; 1.7 x 1011 molecules
each cut
+
with the
same
RE
900 bp; 0.063 µg; 5.7 x 1010 molecules
DNA
ligase
ligation/transformation
• ligation of vector to insert produces several
products
– vector ligated to itself (recircularized)
– insert ligated to itself (circularized, no ori)
– two vectors ligated together
– two (or more) inserts ligated together
– several DNAs ligated together, but not
circularized
– 1 vector ligated to 1 insert DNA
ligation/transformation
• transformation is a very inefficient process
1µg typical plasmid vector = 3 x 1011 copies
added to highly competent E. coli cells
yields
at best
109 antibiotic resistant colonies
3 x 1011/109 = 300 vectors/transformed E. coli
ligation/transformation
• ligation produces a mess of products
• transformation is an inefficient random
process
• selection (antibiotic) sorts out successful
vector transformations
• screening identifies transformants with the
insert in the vector
8.5 x 107 cells are plated
37 form colonies
24
contain
vectors
with
inserts
bacterial transformation has several
potential outcomes
Figure 16.6
creation
of a
DNA library
in
host bacteria using
a
plasmid vector
Figure 16.7
Sources of DNA for Cloning
• chromosomal DNA restriction fragments
– ligated to vectors cut with the same RE
– transferred into bacteria
= a genomic DNA library
• a target DNA is identified by hybridization
reverse
transcription
produces
DNA from
an RNA
template
Figure 16.8
Sources of Genes for Cloning
• mRNAs reverse transcribed into cDNAs
– tissue-specific; age specific; treatment vs.
normal, etc. cDNAs
– ligated to vectors
– grown in host cells and screened by
hybridization
Sources of Genes for Cloning
• make DNA sequences synthetically
– custom oligonucleotides duplicate natural
sequences or create mutant sequences
• site-directed mutagenesis makes an exact
change (mutation) in a cloned gene
What to do With a Cloned (Altered?) Gene
• compare gene expression in two cell types
– a “gene chip” (microarray) displays short
synthetic oligonucleotides
– mRNAs from two different sources are
labeled differently
– mRNAs bind to their complements
– a scanner detects mRNA binding by one cell
type, the other, or both
microarray analysis
compares
gene expression
in
two different
samples
Figure 16.10
What to do With a Cloned (Altered?) Gene
• mutational analysis
– classical genetics found mutations and
studied their effects
– cloning technology causes mutations and
studies their effects
• “knockout” mutations
insertion
of an
inactivated
gene
by
homologous
recombination
Figure 16.9
What to do With a Cloned (Altered?) Gene
• RNA interference (RNAi) produces a
“knockdown” phenotype
– a gene transcribed “backwards” makes an
antisense transcript
• antisense transcript + normal mRNA =
double-stranded RNA
– small interfering RNA (siRNA) forms
double-stranded RNA with normal mRNA
– some viruses inject double-stranded RNA
What to do With a Cloned (Altered?) Gene
• eukaryotic cells attack d.s. RNA
– enzymes “cut” d.s. RNA into 21-23 nt
siRNAs (“dicer”)
– siRNAs guide enzymes to cut target RNAs
(“slicer”)
– siRNAs guide RNA dependent RNA
polymerase to make more d.s. RNA
– [miRNAs control developmental gene
expression]
siRNA is
used to
silence gene
expression
Figure 16.11
What to do With a Cloned (Altered?) Gene
• search for “invisible” interactions
– two hybrid systems identify a receptor’s
ligand
• split a transcription activator into DNAbinding and activating domains
• fuse receptor to DNA-binding domain
• fuse cDNA library to activating domain
• activate a reporter gene when receptor and
ligand bind
a two-hybrid
system detects
binding
proteins
Figure 16.12
What to do With a Cloned (Altered?) Gene
• make the protein…
– a cloning vector tells the cell to replicate it
(with an ori)
– an expression vector tells a cell to efficiently
transcribe and translate a gene in it
an
expression vector
instructs
a
host cell
to
make a protein
Figure 16.13
tissue plasminogen
activator is a clot buster
Figure 16.14
Table 16.1
What to do With a Cloned (Altered?) Gene
• medically useful proteins have been expressed
• plant biotechnology speeds up crop
improvement
– endogenous insecticides
– herbicide resistance
– improved nutrition
– stress tolerance
• “biotech” animals serve as bioreactors to
produce useful proteins
“somatic cell
nuclear transfer”
with
engineered cells
makes
a
sheep
that produces
a useful
protein
CSI
• Short Tandem Repeats (STRs) are used to
identify individuals by “DNA Fingerprinting”
– many sets of STRs exist in the human
genome
– the lengths of STR markers differs for
different individuals
– different-sized STR markers run differently
on agarose gels
DNA
fingerprint
analysis
using an
STR
marker
Figure
16.17
Figure 16.18