WELCOME

BY DIVYA. B I YEAR BIO CHEMISTRY SACW COLEGE

GENE TECHNOLOGY

SPECIFICATION OBJECTIVES

describe and understand the roles of reverse transcriptase, endonucleases and DNA ligase in the manipulation of DNA describe the insertion of DNA into a host cell and the multiplication of the host cell appreciate the use of marker genes to indicate that new genes have been incorporated into host cells understand how protein synthesis is switched on and the synthesis of a new product by the host cell as illustrated by the introduction of new genes into plants using the bacterium Agrobacterium tumefaciens

THE MANIPULATION OF DNA

Gene technology involves the manipulation of genetic material so that genes from one organism can be inserted into the genome of another, unrelated organism. Altered genetic material is referred to as recombinant DNA, and the basis of the technology involves the use of enzymes which enable DNA to be cut, copied and joined.

THE BASIC PRINCIPLES

The isolation of the gene required to produce the product Insertion of this foreign gene into the DNA of a host cell by using a suitable DNA carrier called a vector Checking to find the host cells which contain the new gene Multiplying or cloning the organism containing the new gene to produce large numbers of genetically identical cells or organisms for commercial use

THE ENZYMES INVOLVED

Restriction endonucleases Found naturally in bacteria where they help protect against invasion by viruses by cutting up viral DNA. They are used to cut a gene out of a chromosome . Different restriction enzymes cut at different base sequences because only these bases are the right shape to fit into their active site.

RESTRICTION ENDONUCLEASES

THE ENZYMES INVOLVED

Ligases These enzymes are used to join, or anneal, two strands of DNA. The ligases catalyse the formation of phosphodiester bonds between the pentoses and phosphate groups of two adjacent DNA chains.

THE ENZYMES INVOLVED

Reverse transcriptase An enzyme first isolated from viruses: it will form DNA from an RNA template, allowing complementary, or copy, DNA (cDNA) to be formed from mRNA. This allows double-stranded cDNA sequences to be inserted into a suitable plasmid vector, which can then be used to transform bacterial cells.

CDNA

PLASMIDS

Plasmids are small, circular loops of DNA, which are present in bacterial cells in addition to their circular chromosomal DNA. These small loops of DNA contain some genes, such as genes that confer resistance to antibiotics, and replicate independently of the chromosome. They are used as vectors to introduce genes into a host cell. Once inside the host cell, the plasmid will replicate so that many copies of the original gene will be produced. Plasmids can be cut open by restriction endonucleases, the new gene (which has been cut using the same endonuclease) inserted and then DNA ligase used to join the new gene and the plasmid together.

THE TOOLKIT
The genetic engineer uses five basic tools during the procedure of Recombinant DNA Technology Enzymes capable of cutting DNA at specific sites these enzymes are known as Restriction Enzymes Isolated genes that code for the desired product Vectors (carriers) into which the desired gene may be inserted and which are capable of carrying the gene into a suitable host bacterial plasmids are commonly used as vectors An enzyme capable of glueing an isolated gene into a cut vector DNA ligase is responsible for re-forming the DNA backbone following insertion of the gene A host organism that will take up the vector containing the gene and reproduce rapidly in order to supply many copies of the gene (gene cloning) the bacterium E. coli is commonly used as a host
circular plasmid

plasmid with inserted gene

ISOLATING A GENE USING RESTRICTION ENDONUCLEASE

Restriction Enzymes are the engineers DNA cutting scissors
Restriction enzymes, also known as Restriction Endonucleases, are a group of enzymes found in bacteria that recognise specific DNA sequences of four to six nucleotides and make their incision within that sequence The specific nucleotide sequences, recognised by restriction enzymes, are called restriction sites and these are usually in the form of palindromes Palindromes are nucleotide sequences that are symmetrical, about an axis, and read the same in opposite directions in the two strands of DNA central enzyme cuts axis A restriction enzyme known as portion of G A A T T C Eco R1, makes double-stranded doublestranded cuts between the C T T A A G DNA A and G nucleotides on either side of the central axis enzyme cuts The cuts from this enzyme are staggered and produce single-stranded regions called sticky ends

G C T T

A A A A

C G

sticky ends

BLUNT ENDS
Some restriction enzymes, such as Hpal, cut the DNA strands at positions directly opposite one another, giving blunt ends to the fragments Hpal recognises the nucleotide sequence GTTAAC and cuts between the T and A nucleotides about the central axis
enzyme cuts

G T

A A C T G

C A A T

enzyme cuts

G T

A A C T G
Eco R1 is from Escherichia coli, strain RY13 Bam H1 is from Bacillus amyloliquefaciens, strain H

C A A T
Over seven hundred different blunt ends restriction enzymes have now been identified and isolated from bacterial cells; each enzyme is named after the bacterial strain from which it was derived

THE TOOLKIT
Restriction enzymes that generate sticky ends are very useful tools to the genetic engineer The same restriction enzyme recognises the same nucleotide sequence in the DNA from different species and creates the same sticky ends

When the DNA fragments from the two different species are mixed together, the complementary bases of their sticky ends will be attracted to one another and form hydrogen bonds In this way, DNA fragments from different sources can be brought together and joined DNA ligase is the enzyme that seals fragments of DNA together

Fragment of DNA from species X

G C T T A A

Fragment of DNA from species Y

A A T T C G

Complementary bases on the sticky ends of the DNA from the different species are attracted to one another

Complementary sticky ends created by cutting the DNA from each species with the same restriction enzyme

A A T

C G

C T

A A

Hydrogen bonds form between the bases and the enzyme DNA ligase seals the sugarphosphate backbone of the DNA molecule

G A A C T T

A A G

Recombinant DNA is formed

VECTORS
Vectors are carrier DNA molecules into which DNA fragments containing specific genes can be inserted Vectors are the means by which selected genes are carried into host cells where the desired gene is then cloned The isolated plasmids of bacterial cells and the DNA of bacteriophages (viruses that infect bacteria) are frequently used as vectors Plasmids are small, circular, self-replicating double-stranded DNA molecules found in bacterial cells,which are separate from the main bacterial chromosome

main chromosome

plasmid

BACTERIAL PLASMID
This electron micrograph shows a single bacterial plasmid extracted from the bacterium E. coli

Courtesy of Prof. Stanley Cohen Science Photo Library

Genes coding for desirable products can be spliced into plasmids to form RECOMBINANT PLASMIDS When these plasmids are taken up by bacterial host cells, they replicate along with the host cell and clone the desired gene Plasmids are obtained from cultures of bacterial cells; bacterial cells are broken open and the plasmids are separated out by centrifugation

PLASMIDS

Homogenised bacterial cells, when subjected to centrifugation, provide the plasmids into which foreign genes can be inserted

MAKING RECOMBINANT PLASMIDS

Total human DNA extracted from human cells - known as genomic DNA Plasmid DNA fragment with sticky ends complementary to those on the cut plasmid Both the plasmid and the human DNA are treated with the SAME restriction enzyme so that the DNA from both sources will have complementary sticky ends

The two DNA molecules are attracted to one another and, in the presence of DNA ligase, form a recombinant Recombinant plasmid DNA molecule

When host bacterial cells are mixed with these recombinant plasmids, they may take them up and become transformed; these bacterial cells are now described as transgenic organisms as they contain and express the genetic material from a different species When this transformed bacterial cell divides, the recombinant plasmid replicates and copies of the plasmid (containing the foreign DNA) are passed to the daughter cells

The foreign DNA has been cloned

MANUFACTURING INSULIN

MARKER GENES

Only a few bacteria will take up the plasmid containing the required gene (the recombinant plasmid). These bacteria are said to be modified or transformed. Other bacteria will take up non-recombinant plasmids without the gene. The modified bacteria have to be identified and separated (screened) from the others. This involves genetic markers such as genes for antibiotic resistance.

MARKER GENES
Before

Antibiotic resistant gene

Cut here

After

Human DNA added within the gene

Plasmids with a gene for antibiotic resistance are used as the vectors Cutting the plasmid and inserting the human DNA is inserted within the bases of the antibiotic gene If the gene has been inserted successfully, the antibiotic cannot function properly because it has been damaged

REPLICA PLATING
Replica plating is a technique that allows molecular biologists to transfer samples of bacterial colonies from one nutrient agar plate to another Using this method, duplicate bacterial samples can be grown on a second agar plate in exactly the same position that they were growing on the first, master plate
The felt or velvet-covered tool is pressed gently onto the surface of the first agar plate containing colonies of bacteria

handle

Cells from each of the bacterial colonies stick to the velvet and can be transferred to the replica plate in the same positions relative to one another

sterilised felt or velvet surface

Replica Plating Tool

REPLICA PLATING
These colonies are missing and show where, on the master plate, bacterial colonies containing recombinant plasmids are growing These colonies contain recombinant plasmids; the colonies are removed and grown on a new agar plate

Master plate doesnt contain antibiotic - all bacteria grow

Antibiotic plate transgenic bacteria dont grow

SWITCHING ON GENES

Genes are expressed when their base sequence is being transcribed into mRNA for protein synthesis Many genes have to be activated (switched on) before they can be expressed. This prevents a protein being produced (and wasted) at the wrong time or in the wrong place. Other sections of DNA are involved in this switching on, including promoters, regulators and operators One structural gene (codes for actual functional protein) together with the regulatory genes which control it are known as an operon

SWITCHING ON GENES

THE USE OF THE BACTERIUM AGROBACTERIUM TUMEFACIENS AS A VECTOR WITH PLANTS

This is a bacterium that naturally infects plants causing swellings called galls. To insert a gene into a plant, the required gene is inserted into a plasmid and then the plasmid is inserted into Agrobacterium. Agrobacteruim is then allowed to infect plant cells grown in culture where it transfers plasmids into the plant cells Plants grown from these GM plant cells will contain the inserted gene This was used in 1993 to produce GM oilseed rape plants. The transgenic plants produced a different type of oil which could be extracted and used commercially to make detergents

THE USE OF THE BACTERIUM AGROBACTERIUM TUMEFACIENS AS A VECTOR WITH PLANTS

BY DIVYA. B I YEAR BIO CHEMISTRY SACW COLEGE