Genetic Engineering: Recombinant DNA Technology

The simple addition, deletion, or

manipulation of a single trait in an

organism to create a desired

change.

--- started in1970s

Basic steps in genetic engineering

1. Isolate the gene

2. Insert it in a host using a vector

3. Produce as many copies of the host as

possible

4. Separate and purify the product of the gene

The process of genetic

engineering

Recombinant DNA

Technology in the Synthesis

of Human Insulin

DIABETES and the role of Insulin

• Diabetes mellitus – Greek for “siphon” and Latin for “Honey”

– Characterized by excretion of large amounts of sugar in the urine

– Results from the body’s inability to make sufficient insulin, hormone involved in the regulation of blood sugar (glucose)

– Insulin is secreted into the bloodstream from pancreatic cells where it signals the appropriate tissues (liver and muscle) to remove the excess glucose from the blood.

Types of Diabetes

• Insulin-dependent or Juvenile-onset

– Caused by lack of insulin

– Affects mostly children

• Non-insulin-dependent or adult-onset

– Due to deficient insulin receptors

– Malfunctioning communication system within the body

• Insulin recognizes specific insulin receptors on particular cells and initiates a cascade of reactions that results in the uptake of glucose.

• Regardless of diabetes type, same principle works

– Despite high levels of glucose in the bloodstream, the proper signal does not trigger its uptake

– Individual cells begin to starve even though plenty of glucose is available.

Signalling by insulin

What happens in the absence of Insulin ?

• No glucose uptake

• Cells begin to use fats as primary source of energy.

• Catabolism of fats results in synthesis of ketone bodies,

– Ketone bodies (acetone) are secreted into the bloodstream and function as alternative source of energy for the brain (cannot utilize fat directly)

– Excess of ketone bodies are harmful• Blood becomes acidic

• Toxic at high levels

• Excretion of glucose and ketones in the urine carrying along huge amounts of water and salts, severe dehydration

Reaction of muscle cells

• Muscle cells, requiring large amount of glucose for ATP synthesis, react the starvation by metabolizing proteins

– Large amount of ammonia is produced (toxic to human)

– Normally converted into to urea and excreted

– Ammonia can rise to toxic level under diabetic conditions

A simple breakdown in communication results in greatly altered metabolism in many cells.

The long-term effect of these changes can include kidney failure, heart disease, brain damage, and ultimately death.

Treatment of Diabetes

• Non-insulin-dependent

– Through diet

– Weight reduction

• Insulin-dependent

– Using insulin (serves to bring insulin levels to normal)

– Requires ready supply of insulin

Insulin Production

• Earlier, extracted from pancreas of cows and pigs

• Organ (pancreas) was obtained from slaughterhouses for insulin extraction

• Drawbacks

– Due to increasing incidence of insulin-dependent Diabetes, an increased supply of insulin was required

– Availability of pancreas decreased due to decreased consumption of red meat

• Alternative source of insulin desirable

Genetic engineering for Insulin production

• Creation of genetically engineered bacterial cells that produce human insulin

• Generation of “Bacterial Factories” that can produce cheap, readily available source of insulin

Potential benefits of recombinant insulin

– Ready source of product (bacteria easy to grow)

– No allergic reaction to animal insulin

General considerations

1. Biological reactions inside and outside the cells

– Isolation and purification of functional polymerases

• No interference or contamination from other biological molecules

2. Growing bacteria in the laboratory

• Need to grow bacterial cells without contamination in specific medium according to the requirement

• Broth (to get large quantities of cells)

• Agar (colony formation, to get pure culture and to observe unique properties)

Culture Types

Plate Broth

3. Detecting what happened to individual molecules

– Can we see DNA?

– Gene?

– How will we know that the tube contains DNA?

– How can we say that we are manipulating out desired piece of DNA?

All these issues are of primary importance in genetic engineering

Cloning and expression of Insulin

1. Obtain the gene for insulin from human DNA

2. Insert the gene into bacterial cells

3. Select cells that have desired gene

4. Induce the bacterial cells to express “foreign” gene in

order to produce insulin

5. Collect and purify the final product, insulin

1. Obtaining the insulin gene

• Find the piece of DNA that codes for insulin among the rest of the DNA that makes up human gene

• How?

• The most common method

– Isolate mRNA rather than DNA

• More copies of mRNA than the coding gene itself

• If obtained from pancreas, very high copy number

• Have poly A tail on 3’ end (help in isolation of mRNA)

Conversion into cDNA and amplification of gene

• Reverse Transcription using reverse

transcriptase (RT)

– Synthesizes complementary strand of DNA using

template mRNA (cDNA)

• DNA polymerization using DNA polymerase

– Polymerase chain rection

cDNA synthesis

Polymerase Chain Reaction

Polymerase Chain Reaction

PCR Requirements

1. DNA (purified or a crude extract)

2. Primers specific for the target DNA

3. Free nucleotides (A, G, T, C)

4. DNA polymerase

5. Buffer (containing magnesium)

PCR Primers

• Usually about 18-26 nucleotides in length

• Designed to flank the region to be amplified

• GC content between 50-60 oC

• Melting point determined by G-C and A-T content

– Tm = 4oC (G+C) + 2oC (A+T)

– Ex: a primer with 10 G/C and 10 A/T would have a Tm of

60 oC 4(10) + 2(10)= 60 oC

• Tm of both primers within 2 oC

• Avoid hairpin, dimer and self dimer

2. Inserting genes into bacterial cells

• Can we insert a piece of DNA (PCR amplified) into cell?

1. Linear DNA does not enter the cell easily

2. Bacterial cells do not tolerate DNA that does not form circular structures, linear pieces are destroyed

3. It will not contain the proper signals of transcription, translation and replication systems.

Use of vector for gene insertion

• Genes must be incorporated into vectors (carriers) for safe introduction into bacterial cells

• Vectors are moved between test tube and the cell

– Most common vectors are Plasmids

• Circular pieces of DNA found in different micro-organisms and are replicated independent of the chromosomal DNA

• Usually contains few genes, sometime only one (antibiotic resistance gene)

Basic Properties of Plasmids

• Small, easily manipulated DNA molecules

• Encode genes for antibiotic resistance

• Can be readily transferred into cells and can be isolated easily

• Plasmid contains signals for independent replication within cells.

• Contains multiple but unique cloning sites

DNA inserted into plasmid will be replicated along with the plasmid DNA

Plasmid (Cloning vector)

Tools of the trade

Restriction endonucleases (molecular scissors)

DNA Ligase

Ribonucleases

Terminal transferase

Polynucleotide kinase

Alkaline phosphatase

Restriction endonucleases

• Enzymes that attack and digest internal regions of the DNA of an invading bacteriophage but not that of the host.

• First enzyme extracted from E. coli (cut randomly and not always close to the desired site).

• They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.

• Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragments

• Cleave DNA in a sequence-specific manner

• Most restriction enzymes cut DNA which contains their recognition sequence, no matter what the source of the DNA is.

• Evolved as a defense mechanism against infection by foreign DNA

• Different restriction enzymes in different organisms

Molecular Scissors

Type II RE

3 types of cuts - 5’ overhang, 3’ overhang, blunt

5’-GAATTC-3’3’-CTTAAG-5’

1) 5’ overhang

2) 3’ overhang

3) blunt

5’-GAATTC-3’3’-CTTAAG-5’

5’-CCCGGG-3’3’-GGGCCC-5’

REs as bacterial defense system

• In the bacterial strain EcoR1, the sequence

GAATTC will be methylated at the internal

adenine base (by the EcoR1 methylase).

• The EcoR1 endonuclease within the same

bacteria will not cleave the methylated DNA.

Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own

enzymes

How will you proceed for insertion of gene into vector ?

• No restriction site on the ends of PCR amplified gene

• If there is restriction site ?

– If the sticky ends are compatible ?

– If the ends are incompatible.

• One end is sticky and the other is blunt

• Both ends are sticky but incompatible

Linkers

Adapters

Homopolymer Tailing

Insertion of recombinant DNA into host

• Transformation

– Heat-shock method

– Electroporation

Thanks