Recombinant DNA Technology

1
Chapter 3

• Recombinant DNA Technology
• (genetic engineering)

2
Enzymes that cut and paste DNA
Restriction enzymes cut DNA at specific base
sequences called restriction sites
3
Enzymes that cut DNA are called restriction
enzymes
4
Enzyme DNA ligase enzyme pastes cut ends back
together
5
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
6
Bacterial cells are efficient ways to produce
lots of copies of a foreign gene introduced into
a plasmid
7
Cloning

• Plasmids serve as cloning vectors

• Tumor-inducing
• DNA (Ti plasmid)
• contains 8 tumor-
• inducing genes

transformation

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

8
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

9
Review of molecular biologists toolbox

• Plasmids
• Restriction enzymes
• DNA ligase
• Host bacterial cells to replicate plasmids

10
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

11
Mix plasmid and foreign DNA together with
restriction enzyme and DNA ligase
This plasmid has the lacZ gene inserted
Multiple cloning site inside lacZ gene
(restriction site for insertion site for foreign
gene)
Section of foreign DNA with gene of interest
P
O
Plasmid cloning vector
Foreign DNA
Restriction site
Restriction enzyme
DNA ligase
12
Plasmid cloning vector
Extra-chromosomal DNA carried by bacterial cell
Ampicillin resistance gene ampR (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)
13
Insert plasmid into host bacterial cell for
replication
Bacterial cell
chromosome
14
Cultivate host cell to replicate and produce many
copies of foreign gene
15
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

16
Switching on expression of foreign gene during
cultivation of host bacterial cell
plasmid
No foreign gene inserted
RNA polymerase
chromosome
mRNA
enzyme
colored product
If no foreign gene inserted into restriction
site, then blue colored product is produced
xGal (lactose)
17
If foreign gene is inserted into restriction
site, then no colored product is produced
RNA polymerase
mRNA
no enzyme
no product (no color)
xGal
18
Plating cells on agar surface to promote colony
formation
Visible colony of identical cells
Medium contains ampicillin to allow only the
bacterial cells that contain plasmid with ampR
gene to grow
Semisolid nutrient medium for bacterial cell to
replicate to produce many daughter cells to form
a visible colony
19
Cloning
(restriction sites)
20
(No Transcript)
21
Types of vectors
Maximum insert size (kilobases or kb 1000bp)

• Bacterial plasmid
• bacteriophage
• cosmids
• bacterial artificial chromosome
• yeast artificial chromosome

6-12 25 35 300 200-1000
22
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

Allows bacteria with this plasmid to grow in
presence of ampicillin antibiotic
ori
ampR
lacZ gene
MCS
If plasmid picks up a foreign piece of DNA at the
MCS, then the lacZ gene is non-functional
23
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

24
Making a genomiclibrary
Plate out to form colonies
25
Screening clones forplasmids that pickedup
foreign DNA fragment
26
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

27
stopped
28
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

29
Design primers that specifically target sequences
at the ends of the foreign gene
Plasmid
Foreign gene
30
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
31
Cloning a gene by PCR
Uses a restriction enzyme that recognizes A-T
restriction site for cutting T vector for
insertion of gene
32
T-plasmid vector containing same foreign gene
Host bacterial cell
33
Now, every transformed bacterial cell that
picks up the plasmid contains the same fragment
gene of foreign DNA
34
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
35
Separating DNA fragments produced by treatment
with restriction enzymes
Agarose gel electrophoresis
36
Each band represents a different size fragment
created by cutting the chromosome with a
restriction enzymeDifferent lanes on gel
contain fragments of same DNA cut with different
restriction enzymesWhen you separate DNA
fragments on a gel it is called a Southern gel
Restriction mapping
37
Restriction Mapping
This is the technique used for DNA fingerprinting
Fragment of chromosome
38
Gels that show genes that are being expressed
Gels that reveal mRNA or other types of RNA are
called Northern gels
39
Testing all genes expressed in a tissue quickly
using microarray or gene chip
40
Gene 2
Gene 1
Each spot contains millions of copies of short,
single-stranded DNA-a different gene in each spot
ACCTC
UGGAG
AACTC
41
Computer scans chip and provides a printout of
which genes were expressed
42
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

43
Example of bioinformatics

• Alignment of overlapping sequences
• used to assemble sequence of large pieces of
  DNA (chromosomes)

44
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

45
Comparing the human and mouse genome
46
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

47
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.

48
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

49
Some companies doing this work want to patent the
sequences of fragments of our DNA

• A patent gives legal exclusive right to
  control use
• of sequences contained within fragment

• They see opportunities to turn
• this into a money-making endeavor

• Cost of bringing a new drug (protein) to market
  is about 500 million
• Takes 5-8 years to do this

50
Patents

• Companies want assurances that after investing
  their resources to get a product approved for use
  that another company cant 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

51
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

52
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

53
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

54
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?