Recombinant DNA technology lect

Recombinant DNA Technology


Recombinant DNA technology procedures by

which DNA from different species can be isolated,

cut and spliced together -- new "recombinant "

molecules are then multiplied in quantity in

populations of rapidly dividing cells (e.g. bacteria,

yeast(.


The term gene cloning, recombinant DNA

technology and genetic engineering may seems

similar, however they are different techniques in

Biotechnology and they are interrelated


In the early 1970s it became possible to isolate a

specific piece of DNA out of the millions of base

pairs in a typical genome.


Currently it is relatively easy to cut out a specific

piece of DNA, produce a large number of copies ,

determine its nucleotide sequence, slightly alter it

and then as a final step transfer it back into cell in.


1. DNA molecules are digested with enzymes called

restriction endonucleases which reduces the size of

the fragments Renders them more manageable

for cloning purposes


2. These products of digestion are inserted into a

DNA molecule called a vector Enables desired

fragment to be replicated in cell culture to very high

levels in a given cell (copy (#


3. Introduction of recombinant DNA molecule into

an appropriate host cell

Transformation or transfection

Each cell receiving rDNA = CLONE

May have thousands of copies of rDNA molecules/cell after

DNA replication

As host cell divides, rDNA partitioned into daughter cells


4. Population of cells of a given clone is expanded, and therefore so is the rDNA.

AmplificationDNA can be extracted, purified and used for molecular

analysesInvestigate organization of genes

Structure/functionActivationProcessing

Gene product encoded by that rDNA can be characterized or modified through mutational experiments

Restriction Endonucleases

Endonuclease : Sequence specific nuclease that brak

the nucleic acid chains some where in the interior

rather than atb the ends of the molecules

Econuclease : Nuclease that remove the nucleotides

from the ends of the molecules

A. Origin and function

Bacterial origin = enzymes that cleave foreign DNA Named after the organism from which they were

derivedEcoRI from Escherichia coli

BamHI from Bacillus amyloliquefaciens

Protect bacteria from bacteriophage infectionRestricts viral replication

Bacterium protects it’s own DNA by methylating those specific sequence motifs

Over 200 enzymes identified, many available

commercially from biotechnology companies

B. Classes

Type I & III- Cuts the DNA on both strands but at a non-specific location

at varying distances from the particular sequence that is I : cleave the DNA at site located at 100 bp from the

recognition site III : at about 24 bp

recognized by the restriction enzyme- Therefore random/imprecise cuts

- Not very useful for rDNA applications

Type II

- Cuts both strands of DNA within the particular

sequence restriction site )) recognized by the restriction

enzyme

- Used widely for molecular biology procedures

- DNA sequence = symmetrical

Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence

Some enzymes generate “blunt ends” (cut in middle(

Others generate “sticky ends” (staggered cuts( H-bonding possible with complementary tailsDNA ligase covalently links the two fragments together by

forming phosphodiester bonds of the phosphate-sugar backbones

- EcoRI: Escherichia coli strain R, 1st enzyme - BamHI :Bacillus amyloliquefaciens strain H, 1st

enzymeDiplococcus pneumoniae, 1st enzyme - DpnI :

- HindIII :Haemophilus influenzae, strain D, 3rd enzyme

- BglII :Bacillus globigii, 2nd enzyme - PstI :Providencia stuartii 164, 1st enzyme

- Sau3AI :Staphylococcus aureus strain 3A, 1st enzyme

- KpnI Klebsiella pneumoniae, 1st enzyme


Restriction Enzymes

If two complementary strands of DNA are of equal

length, then they will terminate in a blunt end, as in the following example:

55'-'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3-3''

33'-'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5-5''


Restriction Enzymes

However, if one strand extends beyond the complementary region, then the DNA is said to

possess an overhang:

55'-'-ApTpCpTpGpApCpT-3-3'' 33'-'-TpApGpApCpTpGpApCpTpApCpG-5-5''


Restriction Enzymes

If another DNA fragment exists with a complementary overhang, then these two overhangs will tend to associate with each other and each strand is said to possess a sticky end:


Restriction Enzymes55'-'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT--

33''

33'-'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA--55''

Becomes

55'-'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3-3''

33'-'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5-5''

Recombinant DNA TechnologyDigestion of DNA by EcoRI to produce

cohesive ends ( Fig. 3.1(:

Recombinant DNA TechnologyCreating recombinant DNA :

The first Recombinant DNA molecules were made

by Paul Berg at Stanford University in 1972.

In 1973 Herbert Boyer and Stanley Cohen created the first recombinant DNA organisms.


Creating Recombinant DNA (Fig 3.2(:

Recombinant DNA TechnologySummary of Recombinant DNA technology

process:

Recombinant DNA technology requires DNA extraction, purification, and fragmentation.

Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene.

Recombinant DNA TechnologySummary of Recombinant DNA technology

process:

This portion of the DNA is then coupled to a carrier molecule.

The hybrid DNA is introduced into a chosen cell for reproduction and synthesis.


Transformation and Antibiotic Selection

Transformation is the genetic alteration of a cell

resulting from the introduction, uptake and

expression of foreign DNA.

Recombinant DNA TechnologyTransformation and Antibiotic Selection

There are more aggressive techniques for inserting foreign DNA into eukaryotic cells. For example, through electroporation.

Electroporation involves applying a brief (milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.

Recombinant DNA TechnologyPlasmids and Antibiotic resistance

Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest.

A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a

vector.

Recombinant DNA TechnologyThe gene of interest (resistant to Ampicillin) is

inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that is sensitive to ampicillin.( Text bk:Pg 58(

The bacteria are then spread over a plate that contains ampicillin.


The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate.

Those bacteria which do not acquire the plasmid with the inserted gene of interest will die.


As long as the bacteria grow in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with the gene of interest that has

been inserted to the plasmid .


Fig 3.3 (a(.

Selecting a Gene in a plasmid and Antibiotic selection.

Recombinant DNA TechnologyHuman Gene cloning

Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several

dozen replicas.

Recombinant DNA TechnologyReading materials:

Summary of Recombinant DNA and Cloning (Fig. below(:

Isolation of two kinds of DNA

Treatment of plasmid and foreign DNA with the same restriction enzyme

Mixture of foreign DNA with plasmids

Recombinant DNA TechnologyAddition of DNA ligase

Introduction of recombinant plasmid into bacterial cells

Production of multiple gene copies by gene cloning

Recombinant DNA TechnologySummary of Recombinant DNA and Cloning

(Fig.(:


This segment is "glued" into place using an enzyme called DNA ligase.

The result is an edited, or recombinant, DNA molecule.


Fig: Inserting a

DNA sample into a Plasmid

Vectors for Gene Cloning

The choice of a vector depends on the design of the

experimental system and how the cloned gene will be

screened or utilized subsequently

Most vectors contain a prokaryotic origin of

replication allowing maintenance in bacterial cells.

Some vectors contain an additional eukaryotic origin

of replication allowing autonomous, episomal

replication in eukaryotic cells.

Multiple unique cloning sites are often included for

versatility and easier library construction.

- Antibiotic resistance genes and/or other selectable

markers enable identification of cells that have

acquired the vector construct.

- Some vectors contain inducible or tissue-specific

promoters permitting controlled expression of

introduced genes in transfected cells or transgenic

animals.

- Modern vectors contain multi-functional elements

designed to permit a combination of cloning, DNA

sequencing, in vitro mutagenesis and transcription

and episomal replication.

Main types of vectors

Plasmid, bacteriophage, cosmid, bacterial artificial

chromosome (BAC), yeast artificial chromosome

(YAC), yeast 2 micron plasmid, retrovirus,

baculovirus vector……

. Plasmid vector

- Covalently closed, circular, double stranded DNA

molecules that occur naturally and replicate

extrachromosomally in bacteria

- Many confer drug resistance to bacterial strains

- Origin of replication present (ORI(

-Bacterial cells may contain extrachromosomal DNA called plasmids.

Plasmids are usually represented by small, circular DNA.

Some plasmids are present in multiple copies in the cell

Plasmid vectors are ≈1.2–3kb and contain:

replication origin (ORI) sequence

a gene that permits selection,

Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.

Exogenous DNA can be inserted into the bracketed

region

Origin of replication is a DNA segment recognized by the cellular DNA-

replication enzymes. Without replication

origin, DNA cannot be replicated in the cell

Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endonucleases located next to each other

Restriction sites of the polylinker are not present anywhere else in the plasmid.

Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

Examples

- pBR322

- One of the original plasmids used

- Two selectable markers (Amp and Tet resistance(

- Several unique restriction sites scattered throughout plasmid

(some lie within antibiotic resistance genes = means of screening

for inserts(

- ColE1 ORI

- pUC18

- Derivative of pBR322

- Advantages over pBR322:

- Smaller – so can accommodate larger DNA fragments

during cloning (5-10kbp(

- Higher copy # per cell (500 per cell = 5-10x more than

pBR322(

- Multiple cloning sites clustered in same location =

“polylinker”

. RE DIGESTION OF PLASMID DNA

LIGATION OF DNA SAMPLE AND PLASMID DNA

TRANSFORMATION OF LIGATION PRODUCTS

The process of transferring exogenous DNA into

cells is call “transformation”

There are basically two general methods for

transforming bacteria. The first is a chemical

method utilizing CaCl2 and heat shock to promote

DNA entry into cells.

A second method is called electroporation based

on a short pulse of electric charge to facilitate DNA

uptake.

CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

TRANSFORMATION BY ELECTROPORATION

GROWTH ON AGAR PLATES

TERMS USED IN CLONING

DNA recombination. The DNA fragment to be cloned is inserted into a

vector. Transformation.

The recombinant DNA enters into the host cell and proliferates.

Selective amplification. A specific antibiotic is added to kill E. coli without

any protection. The transformed E. coli is protected by the antibiotic-resistance gene

Isolation of desired DNA clones

CLONING VECTORS

Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.

2. Contain a genetic marker (usually dominant) for selection.

3. Unique restriction sites to facilitate cloning of insert DNA.

4. Minimum amount of nonessential DNA to optimize cloning.

Health & Medicine

Recombinant DNA technology lect