Recombinant DNA technology

Recombinant DNA technology

• Methods used to join together

(recombine) different DNA segments that are not found together in nature.

• This technique is used in genetic analysis to serve several applications:

1. In Vitro Mutagenesis It is much easier to make

mutation in isolated gene than when it is part of a

complex organism structure

2. Study of the gene properties (genotype)

3. Study the expression of gene product (phenotype)

4. Produce large quantity of medically or

agriculturally or industrially important gene product

e.g Insulin production in large quantity by bacteria

Fig. 13-4, p.333

Hydrolysis of DNA by restriction endonucleases

Fig. 13-5, p.334

Production of recombinant DNA

Restriction Endonucleases

(r.e) 1. Enzymes produced by bacteria that hydrolyze

the phosphodiester backbone of DNA at specific sequences

2. The sequences targeted by r.e are palindromes, meaning their sequence reads the same on both strands going in the same direction 3. Most r.e cut DNA in a way that leaves sticky ends that are very useful for recombining DNA from different sources.

Table 13-1, p.335

p.335

Dolly 1996-2003

p.331

In July, 2016, four identical clones of Dolly (Daisy, Debbie, Dianna and Denise) were alive and healthy at nine years old.

The cloning process that produced Dolly

The cloning of a virus

Fig. 13-6, p.336

Fig. 13-7, p.336

The cloning of cells

Fig. 13-8, p.336

The cloning of human DNA fragments with a viral vector

p.338

DNA Plasmid

Extra chromosomal self-replicating genetic elements of a bacterial cell & can be transferred from one strain of a bacterial species to another by cell-to-cell contact.

For A Successful Experiment

When bacteria take up a plasmid, we say they have

been transformed

Bacteria are encouraged to take up foreign DNA by:

1.heat-shock the bacteria at 42 C. followed by placing

them on ice.

2. Place them in an electric field “electroporation”

Then selection through selectable markers on the plasmid.

Fig. 13-11, p.339

A vector cloning site containing multiple restriction sites

Fig. 13-12, p.340

Cloning with pU plasmids

Cloning

1. It refers to creating a genetically identical population

2. DNA can be combined by using r.e that create sticky ends in

the DNA. This rDNA has a target DNA sequence of interest

3. The target DNA sequence is carried in some type of vector,

usually a bacterial plasmid or a virus

4. The target DNA sequence is inserted into a host organism &

the natural doubling time of the organism is used to create

many copies of the target DNA sequence

5. Organisms that are carrying the target DNA are identified

through a process called selection, which often involves antibiotic resistance.

Fig. 13-15, p.345

Synthesis of insulin in humans

Fig. 13-16, p.346

Production of recombinant human insulin

Electroporation. Foreign DNA can be introduced into plant cells by electroporation, the application of intense electric

fields to make their plasma membranes transiently permeable.

Genetic Engineering (g.e)

1. It is the process of inserting genes of interest into

specific organisms for either a medical or scientific

benefit

2. Gene therapy is the process of inserting a missing

gene into an organism

3. Bacteria are often used as the factories to produce a

protein from a cloned gene. This has led to the

production of human insulin & erythropoietin..etc.

4. The gene must be cloned into an expression vector,

usually a plasmid with special features that allows it

to be transcribed & translated in a host cell.

Genetic Engineering in Agriculture

1. Disease resistance e.g. corn & cotton

2. Nitrogen fixation

3. Frost-free plants e.g strawberries & potatoes

4. Tomatoes with a long shef life deactivating the

gene in tomato which produce ethylene.

5. Increased milk production giving cows bovine

somatotropin (BST) “growth hormone”….

6. Good predator attraction straw berry gene on

mustard plants produces a chemical attractant for

predator mites that eat the herbivorous spider mites.

Fig. 13-18, p.349

Transgenic tomato plant : Recombinant DNA methods have produced plants

that resist defoliation by caterpillars, with longer shelf life.

Steps of making recombinant DNA

• Isolation of DNA Cutting DNA in to small

pieces with restriction enzymes Ligate the pieces into cloning vector Transform recombinant DNA molecule into host cell The transformed cell divides many times to form a colony of millions of cells, each carries the recombinant DNA molecule (DNA clone).

1. Isolation of nucleic acids

Is the separation of DNA free from other major molecules such as RNA and proteins, lipids and polysaccharides.

The isolation procedure mainly involve:

DNA isolation from bacteria involves lysis of cell wall by a lysozyme enzyme then alkali denaturation treatment followed by solvents extraction (phenol/chloroform/isoamyl alcohol mixture ) which separates chromosomal DNA from plasmid DNA (remains circular shape and not affected by alkali treatment).

• DNA extraction from human uses blood sample as a source of DNA. In particular, the DNA of white blood cells is isolated in a procedure almost similar to the bacterial DNA without the need for the use of lysozyme enzyme.

2. Cutting DNA

• DNA can be cut into large fragments by

Restriction enzymes (r.e).

• They are group of endonucleases found as protective enzymes in bacteria to destroy foreign DNA in a process called restriction. The host bacterial DNA itself is methylated by a modification enzyme (a methylase) to be protected from the restriction enzyme’s activity.

Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.

The product of restriction enzymes cutting to the DNA is either a sticky (complementary) or blunt(non-complementary) two ends.

In sticky ends: the terminal part of DNA are unequal cohesive single strands which can easily combined together due to complementary property between them.

sticky ends

However , because sticky ends are usually more needed in recombinant DNA

technology than blunt end , an enzyme called Terminal deoxynucleotidyl transferase

can be used to add nucleotides to the blunt-ends of DNA chains to convert them into

sticky ends.

3. Joining of hybrid DNA

By cutting a donor DNA and receiver DNA with the same restriction enzyme, the ends of the two DNA will have the same sticky ends. These two DNAs are mixed in a tube to rejoin together due to the matching of their sticky ends. A small gap will be left that can be sealed by ligase.

4. Amplification of recombinant DNA

I. Cloning of recombinant DNA

It is the method of replicating recombinant DNA inside living cell to generate large population of cells containing identical copies of this type of DNA. The objective of cloning is to replicate recombinant DNA in large amounts, so that it can be used for genetic analysis.

• Since the chemical structure of DNA fundamentally the same in all living organisms, any segment of foreign DNA from an organism is inserted into host DNA of living organism capable of replication, then the foreign DNA will be replicated along with the host cell's DNA during cell division.

Molecular cloning is based on two

basic principles 1.DNA fragments are inserted into plasmid

vectors to produce recombinant DNA

2. The insert-vector recombinant molecules are

transported into living cells, usually E.coli,

which is grown up in colonies to make copies

of the recombinant DNA.

Fig. 13-9, p.338

Selecting for recombinant DNA in a bacterial plasmid

Specific steps of cloning

A. The selection of a vector

B. Generation of foreign DNA fragments (containing

particular gene) and ligate the foreign DNA into vector to

make insert-vector recombinant.

C. The selection of appropriate (competent) E. coli strain

D. Transforming competent E. coli with recombinant vector

E. Grow E. coli colonies to replicate recombinant vectors and

select colonies containing the recombinants DNA.

F. Characterizing the properties of DNA inserts by genetic

analysis.

A. Cloning Vectors

Cloning vector is a plasmid that can be modified to carry new genes, which must have:

• An origin of replication.

• A selectable marker (antibiotic resistance gene, such as ampicillin resistance( ampr) or tetracycline resistance( tetr).

• Multiple cloning site (MCS) a site where insertion of foreign DNA will not disrupt replication or inactivate essential markers.

Plasmid pBR322

Fig. 13-10, p.339

B. Generation and Ligation of Inserts into Vector

The standard procedure for creating the recombinant molecule involves cleaving the

DNA of interest [the insert] and the vector with the same restriction enzyme, followed by incubation with DNA ligase to ligate the insert into the vector.

DNA Library

1. It is a collection of clones of an entire genome

2. The genome is digested with r.e & the pieces are

cloned into vectors & transformed into cell lines

3. Specific radioactive probes to a sequence of

interest are reacted to filters that have copies of

the bacterial colonies in the library. The probe

binds to the sequence of interest, and the colony’s

location can be seen by autoradiography

4. A cDNA library is constructed by using reverse

transcriptase to make DNA from mRNA in a cell.

This cDNA is then used to construct a library similar to a genomic DNA library.

Fig. 13-21, p.352

Formation of cDNA

Fig. 13-19, p.350

Steps involved in the construction of a DNA library

C. Choice of an E. coli host

• Usually an E. coli mutant called lacZΔM15 is used as a

host for the recombinant vector. This mutant is characterized by having inactive β-galactosidase activity due to deletion in the N-terminal part of the enzyme protein coded by defective lacZ gene.

• In the mean time , the defective part of this lacZ gene has been inserted into the vector(plasmid DNA).So, when the plasmid vector is transferred the bacterial host the combined parts complement to each others to give the active enzyme. A test for the formation of active enzyme can be detected by converting a blue colored X-gal dye into white color.

D. Transformation of E. coli with

Recombinant Vector • Transformation is the process of making the

bacteria to take up the recombinant vector

molecule. Nucleic acids do not enter

bacteria under their own power but usually

they require certain procedure involving

treating the bacteria with ice-cold solutions

of CaCl2 followed by short heating to 42

Fig. 13-13, p.341

Clone selection via blue/white screening

Inclusion of ampicillin in the agar allows only bacteria with a plasmid to grow, because these plasmids provide antibiotic resistance

Three types of colonies are produced:

1. Transformed bacteria containing recombinant plasmid

2. Transformed bacteria containing non-recombinant plasmid

3. Non-transformed bacteria

Identification of cells containing plasmids

Inclusion of X-gal allows for blue-white colony screening. Recombinant clones will have white colonies while non-recombinant will have blue colonies.