Lecture Notes on Genetic Engineering

Subject Matter of Genetic Engineering:

DNA is a genetic material which contains all hereditary information needed to create an organism. DNA actually does not make organism, it only makes proteins. DNA is transcribed into mRNA and mRNA is translated into protein and the protein then forms organism.

The specific regions on the DNA molecule that direct the synthesis of proteins are called genes. By changing the nucleotide sequence in DNA, the protein formation is changed.

This leads into either a different protein or inactive protein. Recombinant DNA is the general name for taking a piece of DNA and combining it with another strand of DNA (hence the name recombinant DNA or rDNA). Recombinant DNA is sometimes also referred to as chimera DNA. By combining two or more strands of DNA from two different organisms, scientists are able to create a new strand of DNA.

Recombinant DNA Technology (rDNA Tech) or genetic engineering is concerned with the manipulation of genetic materials towards desired end in a directed way. It is also known as gene cloning. Genetic engineering aims at isolating DNA segments of one organism of interest and fining that with DNA of second unrelated organisms.

In this process the DNA molecules are isolated and cut into pieces by one or more specialised enzymes and the fragments are joined together in a desired combination and restored to a cell for replication and reproduction. Recombinant DNA, thus is a composite DNA molecule that results from the physical combination of DNA segments derived from different sources.

In other words, the Genetic engineering is biochemical manipulation of genes by which foreign genes or the genes of donor organisms are inserted into the DNA or (touted recipient organism through the plasmids of bacteria or through bacteriophages.

Historical Account of Genetic Engineering:

In nature the transfer of genetic material occurs through conjugation, transformation and transduction.

Recombination is brought about by the following two processes:

(i) Natural recombination process, and

(ii) Artificial recombination process.

The natural recombination occurs between closely related species and it does not involve unrelated ones. It occurs during meiosis when similar nucleotide sequences in DNAs of homologous chromosomes come closer, break and exchange segments and then rejoin. Thus a new arrangement of genes results from this recombination.

The artificial recombination process involves unrelated organisms. The genetic material of one organism is inserted into a specialized DNA molecule of another organism giving rise to recombinant or rDNA.

The history of modem genetic engineering goes back to mid 1970s when it became possible to cut DNA into pieces and transfer a particular piece of DNA containing specific information from donor organism to the DNA of the recipient organism.

An year-wise landmarks of the genetic engineering are given here as follows:

1970 Smith and Nathans discovered a new class of enzymes, the restriction enzymes that act as chemical scissors and cut a DNA molecule into smaller fragments.

1972 Berg and others combined DNAs from two viruses to produce what is called recombinant DNA (rDNA).

1973 Cohen and Boyer inserted recombinant DNAs into host bacteria that reproduced or cloned foreign DNAs. With this, the age of genetic engineering begins.

1977 Genetech, one of the first genetic engineering companies starts the biosynthesis of important drugs by rDNA Technology.

1977 Sanger and Gilbert independently discovered techniques for rapid sequencing of nucleotides in DNA molecules.

1982 Human insulin produced by rDNA is marketted under the trade name ‘Humulin’.

1983 Tracy Moreno, a ten year old girl born with growth hormone deficiency, grows 5 inches in one year of treatment with engineered hormones. Human growth hormone genes are inserted into mouse embryo producing a giant mouse. For the first time a human gene functions in another animal.

1997 Scientists remove the DNA-containing nucleus from a female’s egg and replace it with a nucleus from a different animal of the same species. The scientists then place that egg into the uterus of a third animal.

The result, first demonstrated by the birth of a cloned sheep named Dolly, is the birth of an animal that is nearly genetically identical to the animal from which the nucleus was obtained. Such an animal is genetically unrelated to the surrogate mother.

2002 Brigitte Boisselier, a biochemist and director of Clonaid, hinted that human cloning experiments were already underway. She was very, very pleased to announce that the first baby clone was born on December 26, 2002 at 11.55 a.m. Another British baby conceived at the Reproductive Genetic Institute was born on February 9, 2002.

2003 The third cloned baby was born on 3rd January, 2003 to a Dutch woman, the head of Radian sect in Netherlands. Clonaid claims to have produced 13 cloned babies world-wide. Claims of cloned babies have yet to be substantiated primarily because of the risk of incarceration.

In February, 2003 the Human Cloning Prohibition Act was passed which made it a crime for any one, public or private, to conduct somatic cell nuclear transplantation on fertilized or unfertilized human egg cell for reproductive or therapeutic purposes. The penalty for engaging in such research is US $ 1 million fine and a jail term for 10 years.

Cloning is still in its infancy, but it may pave the way for improved domestic animals and medical products.

Procedure of Genetic Engineering:

The gene cloning or recombinant DNA formation requires the following:

1. DNA fragments to be cloned or target gene sequence of desired type.

2. Restriction endonucleases for cutting DNA molecule into fragments.

3. Cloning vector.

4. DNA ligase enzyme for splicing DNA segments.

5. Prokaryotic or Eukaryotic cells to serve as host.

The major steps involved in the genetic engineering are as follows (Fig. 24.1):

1. To break open the living cells:

Several methods are available to break open the living cells. One of the popular methods involves mechanical shearing, the cells in a blender and then treating them with a detergents.

2. Isolation and identification of desired genes or DNA sequence:

The genetic information is stored in DNA. Since the DNA molecules are much longer than all other molecules found in the cells, it has become possible to develop technique of purifying DNA. DNA molecules are spooled on a glass rod. The glass rod bearing the DNA molecules is then lifted out from the mixture of broken cells.

3. Cutting of the DNA molecules into segments containing specific genes from the rest of DNA:

DNA is cut into gene size segments with the help of molecular scissors, called restriction endonucleases. The restriction endonucleases recognise specific base sequences in DNA molecule and make two cuts, one in each strand. Such an action results in generating 3′ hydroxyl and 5′ phosphate terminals.

Several enzymes are used as biological tools in genetic engineering, some of them are as follows:

Exonuclease cleavage:

These enzymes act upon genome and digest the base pairs on 5 or 3 ends of a single stranded DNA

Restriction endonucleases:

Restriction enzymes used in recombinant DNA technology are capable of making cuts in the DNA molecules by two distinct arrangements. One type of cleavage results in blunt end DNA fragments and the other type of cleavage results in cohesive or sticky ended molecule.

The blunt end DNA fragments are formed when the restriction enzymes act on the line of symmetry i.e., they cleave both DNA strands precisely at opposite points (Fig. 24.2A). The cohesive ended molecules are produced when the enzyme acts around the line of symmetry i.e., two strands not cut on opposite points and the cuts are staggered as illustrated below (Fig. 24.2 B).

The sequences recognised by restriction enzymes are often palindromes (i.e., inverts repetitive sequences which are symmetrical.

Restriction endonucleases have the following important features:

a. These enzymes make breaks in palindromic sequences, each consisting of 4-6 complementary bases as illustrated below (Fig. 24.3).

b. The breaks in two DNA strands are usually not directly opposite to each other but are staggered cuts forming cohesive strands.

c. The enzymes yield DNA fragments with complementary ends.

There are two groups of restriction enzymes. Some enzymes recognise a specific nucleotide pair sequence and then cut the DNA at random i.e., at non-specific site away from the recognition site.

The other group of enzymes cleave the DNA at specific site. All restriction enzymes are sequence specific and thus the number of cuts they make in a DNA molecule depends upon the number of times, the specific sequences are repeated in DNA molecules. Different endonucleases found in different organisms recognise different nucleotide sequences and they cut DNA at different cleavage sites.

The restriction enzymes are named according to the name of organisms from which they are derived. The first letter of the genus together with first two letters of speck name of the organism and then first letter of strain in capital form the enzyme name.

Roman number at the end of enzyme name indicates the order of their discovery. Some restriction endonucleases, their source, recognition sequences and sites of cleavage indicated by arrows are presented in Table 24.1.

Since each restriction enzyme recognizes unique base sequence of 4-6 nucleotides irrespective of source of DNA, the pieces obtained from DNA molecule of one organism have the same cohesive ends as the pieces produced by the same enzyme acting on the DNA molecule from another organise.

Since the restriction enzymes recognize unique base sequence, the number of cuts made in DNA molecule from an organism by a particular enzyme is limited.

A typical bacterial DNA molecule which contains around three million base pairs is cut into several hundred to several thousand fragments and nuclear DNA of mammals may be cut into more than a million fragments.

A set of fragment generated by a single restriction enzyme can be detected easily by gel electrophoresis of enzyme treated DNA and particular DNA segments can be isolated by cutting out the portion of the gel containing the fragments and then removing DNA fragment from the gel.

Of the special interest art the smaller DNA molecules such as viral DNA, DNA of Plasmids which may have only 1 – 10 or even none cleavage sites for particular enzyme. Plasmids are especially valuable in recombinant DNA technology because they have single cleavage site for a particular restriction enzyme.

The restriction endonucleases are found in nearly every microorganism, but more in bacteria. No similar enzymes have been identified in a few eukaryotes examined so far. About 975 restriction endonucleases have been isolated from different species of bacteria which recognise more than 100 different nucleotide sequences.

They are now available commercially in pure form and most of these enzymes recognize specific nucleotide sequences. Some restriction enzymes, however, are not specific in their action and they do not cut DNA strands at specific sites. In bacteria these endonucleases act as chemical weapon against invading viruses. If any foreign DNA molecule enters the cell, that it recognized, cut into pieces and made ineffective.

But there is a possibility that the bacterial DNA itself may be cut into pieces by restriction endonucleases which would amount to self-destruction. To prevent this, there is a protective mechanism available in bacterial cytoplasm and in each restriction site one or more nucleotides of corresponding sequences in the genome of bacterium are protected by methylation at an adenine or cytosine residue.

An important event in the study of restriction enzyme with electron microscope was that the fragments produced by many restriction enzymes were spontaneously circularised (Fig. 24.4). The circles could be relinearised by heating but after circularisation if they are treated with DNA ligase, the circularisation becomes stable.

The Eco R₁, enzyme discovered in the laboratory of Dussoix and Boyer was able to cleave a circular DNA molecule to form a linear duplex.

The desired DNA or gene sequence of one organism to be added to the DNA of another organism is called foreign DNA. Foreign DNA may be a fragment of DNA molecule which is enzymatically isolated or a copy DNA or cDNA or ready-made genes procured from gene banks.

Foreign DNA can be procured from a variety of sources depending on the aims and scope of cloning experiment. Copy DNA is synthesized by using messenger RNA as a template with the help of an enzyme reserve transcriptase.

Since identification and characterization of DNA sequences on its : are rather more difficult than using a messenger RNA if it is in pure form complementary DNA or cDNA is synthesized by using mRNA which carries transcript for coding protein. Normally, the flow of genetic information proceeds from DNA —> mRNA —> Protein.

In 1960 Temin and Vallimore independently demonstrated reverse flow of genetic information i.e., complementary DNA or cDNA can be synthesized from single mRNA in presence of enzyme reverse transcriptase. Using this method a particular gene combination is isolated (Fig. 24.5).

c DNA is single stranded DNA (ss DNA). ssDNA is separated from mRNA – DNA complex by alkali hydrolysis. ssDNA is then converted into double stranded DNA (ds DNA) using DNA polymerase of bacterial origin.

The two strands of DNA so obtained are still linked by a covalent bond. The bond is broken by S₁ nuclease in order to make free two DNA strands. Poly A or Poly T tails are now added JKA strands with the help of enzyme terminal transferase in presence of ATP or TTP respectively 24.5). Rapid nucleotide sequencing techniques are now available to use a synthesized gene for cloning.