Recombinant DNA & Biotechnology Chapter 16

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Oct 22, 2013 (3 years and 1 month ago)

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Recombinant DNA

&

Biotechnology

Recombinant DNA


recombinant DNA molecules contain DNA
from different organisms


any two DNAs are joined by
DNA ligase


5’
GGATCATGTA
-
OH

P
-
CCCGATTTCAAT

3’
CCTAGTACAT
-
P

HO
-
GGGCTAAAGTTA



5’
GGATCATGTA
CCCGATTTCAAT


3’
CCTAGTACAT
GGGCTAAAGTTA

DNA ligase

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)


5’GGATC
G
AATT
C
CCGATTTCAAT


3’CCTAG
C
TTAA
G
GGCTAAAGTTA

Eco
RI

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

1/4
6

= 1/4096 x 3x10
9

bp/genome =

7.3 x10
5

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

Y
east
A
rtificial
C
hromosome 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

+

each cut

with the

same RE

DNA

ligase

~4300 bp; 0.1 µg; 1.7 x 10
11

molecules

900 bp; 0.063 µg; 5.7 x 10
10
molecules

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 10
11
copies

added to highly competent
E. coli

cells

yields

at best

10
9

antibiotic resistant colonies


3 x 10
11
/10
9

= 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

37 form colonies

8.5 x 10
7
cells are plated

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
DNA
-
binding

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


S
hort
T
andem
R
epeats (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