chapter 3 recombinant dna technology (genetic engineering)
TRANSCRIPT
Chapter 3
Recombinant DNA Technology
(genetic engineering)
Enzymes that cut and paste DNARestriction enzymes cut DNA at specificbase 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”nducingDNA (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 forforeign 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
PO
This plasmid has the lacZ gene inserted
Plasmid cloning vector
Extra-chromosomal DNAcarried by bacterial cell
Ampicillin resistance gene ampR
(selective marker)
Multiple cloning site inside lacZ gene(restriction site for insertion site forforeign 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 ampR gene to grow
Cloning (restriction sites)
Types of vectors
• Bacterial plasmid
• bacteriophage
• cosmids
• bacterial artificial chromosome
• yeast artificial chromosome
6-12 2535300
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 ampR
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 genomiclibrary
Plate out to
form colonies
Screening clones forplasmids 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
A
C
G
T
G
C
TA
CG
3’ 5’
5’ 3’
CT
CGT
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 rapidapproach to cloningthan making or screening clone libraries.
Makes lots of copiesof foreign genethat is then insertedinto plasmid
Need to know part of sequence of gene
Cloning a gene by PCR
Uses a restriction enzymethat recognizes A-T restrictionsite for cutting T vector for insertion of gene
Host bacterial cell
T-plasmid vector containingsame foreign gene
Now, every “transformed”bacterial cell that picks upthe 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 ofchromosome
This is the techniqueused for DNA fingerprinting
Gels that show genes that are being expressed
Gels that reveal mRNAor other types of RNA are called Northern gels
Testing all genes expressed in a tissue quickly using microarray or “gene chip”
Each spot containsmillions of copies ofshort, single-strandedDNA-a different gene in each spot
Gene 1Gene 2
AACTC
ACCTC
UGGAG
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?