Recombinant DNA technology Barbara McClintock, (1902–1992)

An American scientist and cytogeneticist who was awarded the 1983 Nobel Prize in Physiology Robin Holliday (1932-2014) A British molecular biologist. Holliday described a mechanism of DNA-strand exchange that attempted to explain gene-conversion events that occur during meiosis in fungi.

A Holliday intermediate formed between two bacterial plasmids in vivo, as seen with the electron microscope. Note that such intermediates can form only when the nucleotide sequences of the two parental duplexes are very similar or identical in the region of recombination because specific base pairs must form between the bases of the two parental duplexes.

Recombination

Two DNA molecules can recombine with each other to form new DNA molecules that have segments from both parental molecules.

Classes Of Recombination

1. Homologous genetic recombination

(also called general recombination)

This involves genetic exchanges between any two DNA molecules (or segments of the same molecule) that share an extended region of nearly identical sequence. The actual sequence of bases is irrelevant, as long as it is similar in the two DNAs.

2. Site-specific recombination : The exchanges occur only at a particular

DNA sequence. 3. DNA transposition : It is usually involves a short segment of DNA with the remarkable capacity to move from one location in a chromosome to another. These “jumping genes” were first observed in maize in the 1940s by Barbara McClintock.

Functions of genetic recombination

1. They include roles in specialized DNA repair systems.

2. Specialized activities in DNA replication.

3. Regulation of expression of certain genes.

4. Facilitation of proper chromosome segregation

during eukaryotic cell division.

5. Maintenance of genetic diversity in a population.

6. Implementation of programmed genetic

rearrangements during embryonic development.

DNA recombination

Exchange of DNA which allows mixing of genetic information between gametes that originate from father and mother and produces new combinations of genes. Without this phenomenon, the new gametes will have exactly the same genetic information as the original parents and no genetic variations occur.

Recombination involves the breakage and rejoining of two chromosomes (M and F) to produce two re-

arranged chromosomes (C1 and C2).

Crossing over. (a) Crossing over often produces an exchange of genetic material. (b) The homologous chromosomes of a grasshopper are shown during prophase I of meiosis. Many

points of joining (chiasmata) are evident between the two homologous pairs of chromatids. These chiasmata are the physical manifestation of prior homologous recombination

(crossing over) events.

Recombination of the V and J gene segments of the human IgG kappa light

chain.

The light chain can combine with any of 5,000 possible heavy chains to produce an antibody molecule

Behavior of 2 different genes at different positions on the same chromosome

• When chromosomes go through meiosis, there are

two possible situations:

1. If no cross-over between the two gene loci

occurs (if they are present in short distance from

each other on the same chromosome):

- Alleles segregate together on the same

chromosome - A and B together and a and b

together

2. If there is a cross-over between the two gene loci (when they are present far distance from each other's on the chromosome).

Alleles segregate from each other in Meiosis II

Two recombinant products:

- A and b now together in one meiotic product

- a and B now together in one meiotic product

Two parental products

the other two meiotic products are still AB and ab

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

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.

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

Recombinant DNA – DNA from two

different sources joined together.

1. Cut the DNA and the plasmid using the same restriction enzyme (these enzymes recognize the same base sequences.

2. Insert the foreign DNA into the plasmid.

3. Replace the plasmid into the bacterium

4. Allow the bacterium to reproduce – all future generations have the new DNA

5. Collect the product – it might be insulin or growth hormone, or some other molecule.

Recombinant DNA: Molecule of DNA that is created by joining segments of DNA from different sources.

How to create recombinant DNA:

1. Cut both 2 different samples of DNA with the same restriction enzyme. – Sticky Ends: Single stranded

DNA sequence created after the DNA is cut by certain restriction enzymes

Recombinant DNA

2. Join the 2 cut DNA segments together.

– Since both of the DNA molecules were cut with the same restriction enzymes the sticky ends will contain complimentary bases.

– DNA ligase can be used to fuse together the DNA fragments.

– Beside recombinant DNA, in what other process is DNA ligase used?

Recombinant DNA

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.

p.335

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.

How is recombinant DNA useful?

Recombinant DNA can be inserted into bacterial cells to create human growth hormone.

How to make bacteria with recombinant DNA:

1. Remove a plasmid for a

bacteria cell.

– Plasmid: A small, circular DNA molecule in bacterial cells that is separate from the bacteria’s chromosome.

Recombinant DNA

2. Cut the plasmid and the human DNA with the same restriction enzyme.

3. Use ligase to join the fragment of human DNA containing the insulin gene with the cut bacterial plasmid.

4. Insert the plasmid with recombinant DNA into a bacteria cell.

5. The bacteria cell divides and produces more transgenic bacterial cells that will produce human insulin that can be given to diabetes patients.

Recombinant DNA

Transgenic Organisms: Organisms that have had genes from other species inserted into their genome

Recombinant DNA

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.

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