DNA

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
T G

A C Plasmid DNA is replicated at

same time chromosomal DNA is
replicated

Used to pass genes back and forth

between different bacteria
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) transformation
• contains 8 tumor-
inducing genes

• Use this plasmid

to introduce a
new gene into a
plant
chromosome
 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
Mix plasmid and foreign DNA together with restriction
enzyme and DNA ligase
This plasmid has the lacZ gene inserted

Section of foreign Multiple cloning site inside

DNA with gene of P lacZ gene (restriction site for
interest O insertion site for
foreign gene)
Plasmid
Foreign DNA cloning
vector
Restriction site
Restriction enzyme

DNA ligase
 Plasmid cloning vector
Extra-chromosomal DNA
Ampicillin resistance gene ampR
carried by bacterial cell
(selective marker)

Multiple cloning site

inside lacZ gene
(restriction site for
insertion site for
foreign gene)
Foreign
gene
inserted lacZ gene with promoter
(used to switch in
expression of foreign
gene when inside a
bacterial host cell)
Insert plasmid into host bacterial
cell for replication

chromosome

Bacterial cell
 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
plasmid No foreign gene inserted

RNA polymerase
chromosome

mRNA

enzyme

colored product
If no foreign gene inserted
xGal (lactose)
into restriction site, then blue
colored product is produced
If foreign gene is inserted into restriction site, then no
colored product is produced

RNA polymerase

mRNA

no enzyme

xGal no product (no color)

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
Cloning (restriction sites)
 Types of vectors
Maximum insert size
(kilobases or kb [1000bp])
• Bacterial plasmid 6-12
• bacteriophage 25
• cosmids 35
• bacterial artificial 300
chromosome
• yeast artificial 200-1000
chromosome
 • origin of Practical Features of DNA
Cloning Vectors (Plasmids)
replication (ori)
• multiple cloning Allows bacteria with this
plasmid to grow in presence
sites (MCS) or of ampicillin antibiotic
restriction sites ori ampR
• selectable
lacZ gene
markers
• RNA polymerase
MCS
promoter
sequences
• DNA sequencing
primer sequences
If plasmid picks up a foreign piece of DNA at the MCS, then the lacZ gene is non-functional
 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
 Design primers that specifically target
sequences at the ends of the foreign gene

Plasmid
5’ 3’ Reverse
T primer
A T
G
C G
C G
DNA polymerase
C
Foreign gene

Forward
T A
primer T
C G C

3’ 5’
 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
T-plasmid vector containing
same foreign gene

Host bacterial cell

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 bands
DNA by
electrophoresis
Separating DNA fragments produced by
treatment with restriction enzymes
Agarose gel electrophoresis
Each band represents a Restriction mapping
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

This is the technique

used for DNA
fingerprinting

Fragment of
chromosome
 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”
 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