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

If no cross-over between the two gene loci Behavior of 2 different genes at different positions on the same chromosome When chromosomes go through meiosis, there are two possible situations: 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.

Hydrolysis of DNA by restriction endonucleases FIGURE 13.4 Hydrolysis of DNA by restriction endonucleases. (a) Separation of ends. (b) Resealing of ends by DNA ligase. Fig. 13-4, p.333

Production of recombinant DNA FIGURE 13.5 (1) Foreign DNA sequences can be inserted into plasmid vectors by opening the circular plasmid with a restriction endonuclease. (2) The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. Fig. 13-5, p.334

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. DNA plasmids—extrachromosomal selfreplicating genetic elements of a bacterial cell. p.338

Restriction Endonucleases (r.e) Enzymes produced by bacteria that hydrolyze the phosphodiester backbone of DNA at specific sequences 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. Cut the DNA and the plasmid using the same restriction enzyme (these enzymes recognize the same base sequences. Insert the foreign DNA into the plasmid. Replace the plasmid into the bacterium Allow the bacterium to reproduce – all future generations have the new DNA Collect the product – it might be insulin or growth hormone, or some other molecule.

Recombinant DNA Recombinant DNA: Molecule of DNA that is created by joining segments of DNA from different sources. How to create recombinant DNA: 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 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?

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 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.

Methylation of endogenous DNA protects it from cleavage by its own restriction endonucleases.

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.

Recombinant DNA 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: 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 Cut the plasmid and the human DNA with the same restriction enzyme. Use ligase to join the fragment of human DNA containing the insulin gene with the cut bacterial plasmid. Insert the plasmid with recombinant DNA into a bacteria cell. 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

Synthesis of insulin in humans FIGURE 13.15 Synthesis of insulin in humans. The insulin gene is a split gene. The intervening sequence (intron) encodes an RNA transcript that is spliced out of the mRNA. Only the portions of the gene called exons are reflected in the base sequence of mRNA. Once protein synthesis takes place, the polypeptide is folded, cut, and spliced. The end product, active insulin, has two polypeptide chains as a result. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-15, p.345

Production of recombinant human insulin FIGURE 13.16 Active human insulin can be produced in bacteria by the use of two separate batches of E. coli. Each batch produces one of the two chains, the A chain or the B chain. The two chains are mixed to produce active insulin. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-16, p.346

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.

Selecting for recombinant DNA in a bacterial plasmid FIGURE 13.9 Selecting for recombinant DNA in a bacterial plasmid. The plasmid also contains a gene for antibiotic resistance. When bacteria are grown in a medium that contains the antibiotic, those that have acquired a plasmid will grow. Bacteria without a plasmid cannot grow in this medium. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-9, p.338