Chapter 3

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14 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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Chapter 3

Recombinant DNA Technology

(genetic engineering)

Enzymes that cut and paste DNA

Restriction enzymes

cut DNA at specific

base sequences called
restriction sites

Enzymes that cut DNA are called
restriction
enzymes

Enzyme DNA ligase

enzyme pastes cut ends back together

Cloning
: the introduction of new or foreign
genes into plasmids and other “vectors”

This is when scientists take control of the natural
processes that the bacteria have evolved to
promote exchange of genes between individuals
of the same or different “species”

Circular extrachromosomal DNA
found commonly in bacteria


Plasmid DNA is replicated at same
time chromosomal DNA is
replicated


Used to pass genes back and forth
between different bacteria

A

T

C

G

Bacterial cells are efficient ways to produce lots of
copies of a foreign gene introduced into a plasmid

Cloning


Plasmids serve as
cloning
vectors



T
”umor
-

i
”nducing

DNA (
Ti plasmid
)


contains 8 tumor
-

inducing genes


Use this plasmid
to introduce a new
gene into a plant
chromosome

transformation

Concerns about cloning


What might happen if cloned bacteria were
to leave the lab and transfer their genes to
other bacteria or even humans?


E. coli was initially the most common host for
these cloned genes


Benefits and hazards discussed in 1975 at a
meeting


National Institutes of Health (NIH) formed the
Recombinant DNA Advisory Committee
(RAC)


Guidelines established for recombinant DNA
research by scientific community

Review of molecular biologists’
toolbox


Plasmids


Restriction enzymes


DNA ligase


Host bacterial cells to replicate plasmids


Recombinant DNA technology has become a
way for geneticists to express genes from other
organisms in bacteria



Human
insulin gene
was cloned into a bacterial plasmid


and expressed (gene mRNA protein) in a bacterium in
1977.

-
Cheap and pure source of insulin


Humulin

growth hormone was first recombinant


DNA product to be approved by FDA in 1992


Currently over 100 products on market produced by


recombinant techniques

Multiple cloning site inside
lacZ gene
(restriction site for
insertion site for

foreign gene)

Foreign DNA

Section of foreign
DNA with gene of
interest

Plasmid
cloning
vector

Mix plasmid and foreign DNA together with restriction
enzyme and DNA ligase

Restriction enzyme

DNA ligase

Restriction site

P

O

This plasmid has the lacZ gene inserted

Plasmid cloning vector

Extra
-
chromosomal DNA

carried by bacterial cell

Ampicillin resistance gene
amp
R


(selective marker)

Multiple cloning site
inside
lacZ gene

(restriction site for
insertion site for

foreign gene)


lacZ

gene

with promoter
(used to switch in
expression of foreign gene
when inside a bacterial
host cell)

Foreign
gene
inserted

Insert plasmid into host bacterial
cell for replication







Bacterial cell

chromosome

Cultivate host cell to replicate and
produce many copies of foreign gene

Bacterial cell

Detecting cells that have foreign gene
inserted in lacZ gene on plasmid


Need some way to check to see that foreign
gene was inserted into the plasmid so when
you cultivate the cell, you know you are
producing more copies of foreign gene

Switching on expression of foreign gene
during cultivation of host bacterial cell

xGal (lactose)

RNA polymerase

colored product

enzyme

mRNA

If no foreign gene inserted
into restriction site, then blue
colored product is produced

plasmid

chromosome

No foreign gene inserted

RNA polymerase

no product (no color)


no enzyme

mRNA

xGal

If foreign gene is inserted into restriction site, then no
colored product is produced

Plating cells on agar surface to
promote colony formation

Semisolid nutrient medium for bacterial cell to
replicate to produce many daughter cells to form a
visible colony

Visible colony of
identical cells

Medium contains
ampicillin to allow only
the bacterial cells that
contain plasmid with amp
R

gene to grow

Cloning

(restriction sites)

Types of vectors


Bacterial plasmid


bacteriophage


cosmids


bacterial artificial
chromosome


yeast artificial
chromosome

6
-
12


25

35

300


200
-
1000

Maximum insert size

(kilobases or kb [1000bp])

Practical Features of DNA
Cloning Vectors (Plasmids)


origin of
replication (ori)


multiple cloning
sites (MCS) or
restriction sites


selectable
markers



RNA polymerase
promoter
sequences


DNA sequencing
primer sequences

ori

amp
R

MCS

lacZ gene

If plasmid picks up a foreign piece of DNA at the MCS, then the lacZ gene is non
-
functional

Allows bacteria with this
plasmid to grow in presence
of ampicillin antibiotic

You can use plasmids to create a
clone “library”

Purpose: To distribute different sections of a DNA
molecule or chromosome into a vector that allows
the genes contained in the section to be
characterized

Making a genomic

library

Plate out to


form colonies

Screening clones for

plasmids that picked

up foreign DNA fragment

What if you know a part of the
base sequence of the gene you
are looking for?

The Human Genome Project has
given us this information for all the
genes in our chromosomes

stopped

Polymerase chain reaction (PCR)


Has revolutionized molecular biology and
biotechnology.


Most useful when you know at least some
of the base sequence of the gene you are
interested in


Only need to know a sequence containing
10
-
20 base pairs in a gene that may contain
thousands of base pairs

3’ 5’

5’ 3’

C

T

C

G

T

DNA polymerase

Forward
primer

Reverse
primer

Design primers that specifically target
sequences at the ends of the foreign gene

Foreign gene

Plasmid

Polymerase Chain
Reaction (PCR)

Much more rapid

approach to cloning

than making or

screening clone

libraries.


Makes lots of copies

of foreign gene

that is then inserted

into plasmid


Need to know part of
sequence of gene

Cloning a
gene by PCR

Uses a restriction enzyme

that recognizes A
-
T restriction

site for cutting T vector for

insertion of gene

Host bacterial cell

T
-
plasmid vector containing

same foreign gene

Now, every “transformed”

bacterial cell that picks up

the plasmid contains the

same fragment “gene” of

foreign DNA

How do you recover foreign DNA
fragment containing gene of interest?


Pellet cells from
culture medium


Resuspend cells in
solution that breaks up
“lyses” cells to release
DNA


Separate host cell
DNA from plasmid
DNA by
electrophoresis

DNA bands

Separating DNA fragments produced by
treatment with restriction enzymes

Agarose gel electrophoresis

Each band represents a
different size fragment
created by cutting the
chromosome with a
restriction enzyme


Different lanes on gel
contain fragments of same
DNA cut with different
restriction enzymes


When you separate DNA
fragments on a gel it is
called a
Southern gel

Restriction mapping

Restriction Mapping

Fragment of

chromosome

This is the technique

used for DNA
fingerprinting

Gels that show genes that are
being expressed

Gels that reveal mRNA

or other types of RNA

are called
Northern gels

Testing all genes expressed in a tissue
quickly using microarray or “gene chip”

Each spot contains

millions of copies of

short, single
-
stranded

DNA
-
a different gene in
each spot

Gene 1

Gene 2

A
A
C
T
C

A
C
C
T
C

U
G
G
A
G

Computer scans chip and provides a
printout of which genes were expressed

Bioinformatics


Database manipulation of DNA sequence
information


Application of computer science and
information technology to help understand
biological processes


Use of computers to relate gene sequence to
protein structure and function


Example of bioinformatics

Alignment of
overlapping sequences



used to assemble
sequence of large
pieces of DNA
(chromosomes)

Using Bioinformatics


GenBank
-
a library of base sequences that
have been catalogued


www.ncbi.nlm.nih.gov/blast


useful for matching your sequences from your clone
library with sequences found and deposited by
others previously


go to blastn


type in
AATAAGAACCAGGAGTGGA


BLAST finds the match to your sequence to be the gene
for early
-
onset breast cancer, BRCA
-
1


each unique sequence is assigned an accession
number to make it easy for scientists to refer back to
that sequence

Comparing the human and mouse
genome

More things you can do


www.ncbi.nlm.nih.gov/Omim


search Omim database


type in a word for a disease then search


the database provides you with a list of diabetes
-
related genes


click on one
-
it provides you with all types of
information on these genes


click on gene map


click on IDDM1

»
click on 6p21.3

»
it shows you the locus on the chromosome where the
gene resides (find 222100)

»
click on 222100
-
it verifies that you have located the
gene of interest

Search for a gene you are
interested in


www.ncbi.nlm.nih.gov/disease


lists different metabolism along left


at top “click here” takes you to all the
chromosomes


click on chromosome 7


gives you more info on the genes on that
chromosome


shows you where the genes for different diseases are
located on that chromosome.


Summary


Restriction sites and enzymes


Cloning vectors (plasmids)


Inserting foreign genes in plasmids


Hosts cells for replicating plasmids (bacteria)


Clone libraries, cDNA libraries


Screening for recombinant plasmids


Polymerase chain reaction (PCR)


Reverse transcription PCR for detecting mRNA


Separating DNA fragments on gels


Gene chips


Bioinformatics

Some companies doing this work want to
patent

the sequences of fragments of our DNA


Cost of bringing a new drug (protein) to
market is about $500 million


Takes 5
-
8 years to do this






They see opportunities to turn


this into a money
-
making endeavor



A patent gives legal exclusive right to control use


of sequences contained within fragment

Patents


Companies want assurances that after
investing their resources to get a product
approved for use that another company
can’t come in and make $$ without such an
investment


Since 1980, the U.S. Patent Office has
awarded patents on more than 20,000 gene
sequences


Patent process


3 categories


products or composition of matter


methods of use


manufacturing processes


Conditions that must be met to receive a patent


must be new (not previously published or described)


must be useful


not obvious to one skilled in the field

Elements of a patent application


Description of technical field to which invention
applies


Description of problems to be solved and prior
“art”


How the invention improves upon prior art


Summary enumerating fundamental components
of invention


Description of invention and indispensable steps
for constructing invention

Elements of a patent application


Claims that outline the elements to be protected
by law.


A claim cannot be so broad that it infringes upon
prior “art”


A claim should not be so narrowly focused that the
applicant could risk losing property claims


Patent attorneys are skilled in preparing patent
application


Upon review of application by U.S. Patent
Office, a decision will be made whether a new
patent is justified
-
if so, a patent no. is assigned

New Patent Issues


Sequences may or may not encode a gene


Sequence may control regulation of nearby
genes.


Many scientists believe patenting should be
reserved for the new technology used to
discover genes and their functions and their
application rather than the sequence.


Is it ethical to patent a sequence?


What are the possible consequences?