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?