Chapter 23 (Part 1)

Recombinant DNA Technology

Recombinant DNA Technology

• Methods for isolating, manipulating, and amplifying identifiable DNA sequences.

• Allows us to study the structure and function of individual genes.

• Allows for the directed genetic manipulation of organism (modify gene function, insert novel genes)

Cloning• Clone: a collection of molecules or

cells, all identical to an original molecule or cell

• To "clone a gene" is to make many copies of it - for example, in a population of bacteria

• Gene can be an exact copy of a natural gene

• Gene can be an altered version of a natural gene

• Recombinant DNA technology makes it possible

• Allows for in vitro manipulation of a individual gene

Tools Needed for Cloning(Think of it as a cutting and pasting

process)

• cDNA or genomic library (source of DNA to cut)

• Plasmid (where you want to paste it)• Restriction enzymes (scissors)• DNA ligase (paste)• E. coli (biological machine needed to

amplify DNA)

Plasmids• Naturally occurring

extrachromosomal DNA• Self replicating circular double

stranded DNA molecules that have their own origin of replication

• Usually present in multiple copies per cell

• Plasmids can be cleaved by restriction enzymes, leaving sticky ends

• Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

Cloning Vector

Required features1. Origin of

replication2. Selectable marker3. Screenable marker

for recombinant molecules

4. Cloning sites

Restriction Enzymes• Bacteria protect themselves from

attack by viruses and other bacteria using a restriction/modification system.

• Allows bacteria to recognize and destroy foreign DNA

• Bacteria contain DNA methylases that modify their chromosomal DNA at specific sequences.

• Also contain restriction endonucleases that recognize and cleave these same sequences when they are not methylated

Restriction Modification System

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA* *

AAGATGCGAATTCGTACA* *

DNA methylase

Restriction endonuclease

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA

AAGATGCG AATTCGTACA

DNA methylase

Restriction endonuclease

Restriction Enzymes

• Type I – Contain methylase and endonulcease fuctions. Require ATP for hydrolysis and S-adenosylmethionine for methylation

• Type II – contain only endonulcease function,. Does not require ATP for hydrolysis.

• Both types recognize palindrome sequences (sequences that read the same if read forward or backwards – e.g. “BOB” or “DEED”

Type II Restriction Enzymes

• Names use 3-letter italicized code:

• 1st letter - genus; 2nd,3rd - species

• Following letter denotes strain • EcoRI is the first restriction

enzyme found in the R strain of E. coli

5’ ATGCGAATTCCGGTT 3’3’ TACGCTTAAGGCCTT 5’

5’-ATGCG-3’ 5’-AATTCCGGTT-3’3’-TACGCTTAA-5’ 3’-GGCCTT-5’

EcoR1

5’ ATGCGATATCCGGTT 3’3’ TACGCTATAGGCCTT 5’

5’-ATGCGAT-3’ 5’-ATCCGGTT-3’3’-TACGCTA-5’ 3’-TAGGCCTT-5’

EcoRV

Sticky-end cutter

Blunt-end cutter

Restriction Enzymes

• Restriction enzymes can recognize specific 4 base, 6 base, 8 base sequences.

• The probability that a given piece of DNA will contain a specific restriction site is = n4

• n = the number of bases in the restriction site• So for a 6 base cutter (64), you would expect to

find your site every ~1300 base pairs. So in a 10,000 bp fragment there is likely to by 7 or 8 restriction sites corresponding to your enzyme.

• You can characterize DNA fragments using gel electrophoresis

T4 DNA Ligase

Transformation• All of the previous steps were

performed in vitro. • We have generated a very small

amount of a recombinant plasmid

• Need to amplify in bacteria to get enough to work with.

• Transformation – process to mobilize DNA into bacterial host

• Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid

How to produce a recombinant protein

10 to 70% of cellular protein

0.1 to 1% of

cellular protein

Cloning a gene from a DNA libraries

• Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell

• Can't isolate it directly • Trick is to find the fragment or

fragments in the library that contains the desired gene

cDNA

cDNA Library

cDNA

Library Screening• DNA probe hydridization• Requires that you know the protein or amino acid

sequence of the gene of interest.• Need to denature (make single stranded) and

immobilize the DNA from each clone of the library to a filter (nitrocellulose or nylon)

• Make a labeled single stranded DNA/RNA probe (can use radioactive of fluorescent analogous of specific nucleotide triphosphates)

• Labeled single stranded DNA/RNA fragments will base pair (hydridize) with the target DNA on the filter

• Identify clones that are labeled.

DNA hydridization screening for specific gene

•Requires that you know something about the gene sequence•Can get sequence information form purified protein

Now that we have the gene, what do we do with

it?• We could use it make a lot of

protein in a microbial protein expression system

• We could use it to genetically manipulate organisms

• We could use it as a diagnostic tool

Why use recombinant Proteins?

• Proteins are often only available in small amounts in a given tissue

• Tissue sources may not be readily available

• It is time consuming and expensive to purify protein from tissues

• It is difficult to obtain absolutely pure protein

Insulin• Was first purified from human

pancreas from cadavers and then from pig pancreas.

• Genentec expressed insulin gene in microbial host

• Can grow microbes in large fermenters to produce unlimited supply of insulin.

Product name  Protein type Application Company

Adagen (Adenosine

deaminase )

An enzyme Severe combined immunodeficiency disease (SCID)

Enzon

Genotropin (Recombinant

growth hormone)

A hormone Growth hormone deficiency (GHD)

in children

Pharmacia & Upjohn

Humalog (Recombinant

human insulin)

A hormone Diabetes Eli Lilly

Nabi-HB (Anti-Hepatitis B)

 An antibody Hepatitis-B  Nabi

Novo Seven (Recombinant coagulation factor VIIa)

A modified factor Hemophillia patients with

inhibitors

Novo Nordisk

Ontak (Diphtheria

toxin-interleukin-2)

A fusion protein Cutaneous T-cell lymphoma (CTCL)

Ligand Pharmaceuticals

Roferon-A (Recombinant

interferon alfa-2a)

A modifier Hairy cell leukemia or AIDS-related

Kaposi's sarcoma

Hoffmann-La Roche

Recombinant proteins are also important to

research• For enzyme analysis need pure

protein• For structural analysis need

lots (milligram amounts) of very pure protein

• Need pure proteins to make diagnostic tools such as antibodies

Genetic Modification of Higher Organisms

• Can introduce gene into animals and plants

• These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc….

• Has also been used to develop improved agricultural products

Genetically Engineered SalmonIs Bigger Better?

http://www.agwest.sk.ca/sabic_index_tp.shtml

Plant Genetic EngineeringImproved Agricultural Production

A. Herbicide Resistance

B. Pest Resistance

Improved Nutrition

A. Vitamins - Golden Rice, Vitamin E

B. Increase essential Amino Acid Content

Chemical Synthesis

A. Bio-plastics

B. Bio-diesel

C. Lubricants/detergents

D. Rubber

GMO Concerns

• Ecological Concern

• Potential Food Allergens

• Antibiotic Resistance

GMO Benefits

• Lower application of herbicides and pesticides

• Creation of foods with increased nutrition

• Creation of bio-based alternative to petroleum based products

http://www.colostate.edu/programs/lifesciences/TransgenicCrops/