Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology • Cleaving and Rejoining DNA • Getting New Genes into Cells • Sources of Genes for Cloning • Some Additional Tools for DNA Manipulation • Biotechnology: Applications of DNA Manipulation

Cleaving and Rejoining DNA • Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources. • Recombinant DNA technology uses the techniques of sequencing, rejoining, amplifying, and locating DNA fragments, making use of complementary base pairing.

Cleaving and Rejoining DNA • Bacteria defend themselves against invasion by viruses by producing restriction enzymes which catalyze the cleavage of DNA into small fragments. • The enzymes cut the bonds between the 3¢ hydroxyl of one nucleotide, and the 5¢ phosphate of the next. • There are many such enzymes, each of which recognizes and cuts a specific sequence of bases, called a recognition sequence or restriction site (4 to 6 base pairs long).

Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes

Cleaving and Rejoining DNA • Host DNA is not damaged due to methylation of certain bases at the restriction sites; this is performed by enzymes called methylases. • The enzyme EcoRI cuts DNA with the following paired sequence: • 5¢ ... GAATTC ... 3¢ • 3¢ ... CTTAAG ... 5¢ • Notice that the sequence is palindromic: It reads the same in the 5¢-to-3¢ direction on both strands.

Cleaving and Rejoining DNA • Using EcoRI on a long stretch of DNA would create fragments with an average length of 4,098 bases. • Using EcoRI to cut up small viral genomes may result in only a few fragments. • For a eukaryotic genome with tens of millions of base pairs, the number of fragments will be very large. • Hundreds of restriction enzymes have been purified from various organisms, and these enzymes serve as “knives” for genetic surgery.

Cleaving and Rejoining DNA • The fragments of DNA can be separated using gel electrophoresis. Because of its phosphate groups, DNA is negatively charged at neutral pH. • When DNA is placed in a semisolid gel and an electric field is applied, the DNA molecules migrate toward the positive pole. • Smaller molecules can migrate more quickly through the porous gel than larger ones. • After a fixed time, the separated molecules can then be stained with a fluorescent dye and examined under ultraviolet light.

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Cleaving and Rejoining DNA • Electrophoresis gives two types of information: • Size of the DNA fragments can be determined by comparison to DNA fragments of known size added to the gel as a reference. • A specific DNA sequence can be determined by using a complementary labeled single-stranded DNA probe. • The specific fragment can be cut out as a lump of gel and removed by diffusion into a small volume of water.

Figure 16.3 Analyzing DNA Fragments

Cleaving and Rejoining DNA • Some restriction enzymes cut DNA strands and leave staggered ends of single-stranded DNA, or “sticky” ends, that attract complementary sequences. • If two different DNAs are cut so each has sticky ends, fragments with complementary sticky ends can be recombined and sealed with the enzyme DNA ligase. • These simple techniques, which give scientists the power to manipulate genetic material, have revolutionized biological science in the past 30 years.

Figure 16.4 Cutting and Splicing DNA

Getting New Genes into Cells • The goal of recombinant DNA work is to produce many copies (clones) of a particular gene. • To make protein, the genes must be introduced, or transfected, into a host cell. • The host cells or organisms, referred to as transgenic, are transfected with DNA under special conditions. • The cells that get the DNA are distinguished from those that do not by means of genetic markers, called reporter genes.

Getting New Genes into Cells • Bacteria have been useful as hosts for recombinant DNA. • Bacteria are easy to manipulate, and they grow and divide quickly. • They have genetic markers that make it easy to select or screen for insertion. • They have been intensely studied and much of their molecular biology is known.

Getting New Genes into Cells • Bacteria have some disadvantages as well. • Bacteria lack splicing machinery to excise introns. • Protein modifications, such as glycosylation and phosphorylation, fail to occur as they would in a eukaryotic cell. • In some applications, the expression of the new gene in a eukaryote (the creation of a transgenic organism) is the desired outcome.

Getting New Genes into Cells • Saccharomyces, baker’s and brewer’s yeast, are commonly used eukaryotic hosts for recombinant DNA studies. • In comparison to many other eukaryotic cells, yeasts divide quickly, they are easy to grow, and have relatively small genomes (about 20 million base pairs).

Getting New Genes into Cells • Plants are also used as hosts if the goal is to make a transgenic plant. • It is relatively easy to regenerate an entire plant from differentiated plant cells because of plant cell totipotency. • The transgenic plant can then reproduce naturally in the field and will carry and express the gene on the recombinant DNA.

Getting New Genes into Cells • New DNA can be introduced into the cell’s genome by integration into a chromosome of the host cell. • If the new DNA is to be replicated, it must become part of a segment of DNA that contains an origin of replication called a replicon, or replication unit.

Getting New Genes into Cells • New DNA can be incorporated into the host cell by a vector, which should have four characteristics: • The ability to replicate independently in the host cell • A recognition sequence for a restriction enzyme, permitting it to form recombinant DNA • A reporter gene that will announce its presence in the host cell • A small size in comparison to host chromosomes

Getting New Genes into Cells • Plasmids are ideal vectors for the introduction of recombinant DNA into bacteria. • A plasmid is small and can divide separately from the host’s chromosome. • They often have just one restriction site, if any, for a given restriction enzyme. • Cutting the plasmid at one site makes it a linear molecule with sticky ends. • If another DNA is cut with the same enzyme, it is possible to insert the DNA into the plasmid. • Plasmids often contain antibiotic resistance genes, which serve as genetic markers.

Figure 16.5 (a) Vectors for Carrying DNA into Cells

Getting New Genes into Cells • Only about 10,000 base pairs can be inserted into plasmid DNA, so for most eukaryotic genes a vector that accommodates larger DNA inserts is needed. • For inserting larger DNA sequences, viruses are often used as vectors. • If the genes that cause death and lysis in E. coli are eliminated, the bacteriophage l can still infect the host and inject its DNA. • The deleted 20,000 base pairs can be replaced by DNA from another organism, creating recombinant viral DNA.

Getting New Genes into Cells • Bacterial plasmids are not good vectors for yeast hosts because prokaryotic and eukaryotic DNA sequences use different origins of replication. • A yeast artificial chromosome, or YAC, has been made that has a yeast origin of replication, a centromere sequence, and telomeres, making it a true eukaryotic chromosome. • YACs have been engineered to include specialized single restriction sites and selectable markers. • YACs can accommodate up to 1.5 million base pairs of inserted DNA.

Figure 16.5 (b) Vectors for Carrying DNA into Cells

Getting New Genes into Cells • Plasmid vectors for plants include a plasmid found in the Agrobacterium tumefaciens bacterium, which causes the tumor-producing disease, crown gall, in plants. • Part of the tumor-inducing (Ti) plasmid of A. tumefaciens is T DNA, a transposon, which inserts copies of itself into the host chromosomes. • If T DNA is replaced with the new DNA, the plasmid no longer produces tumors, but the transposon still can be inserted into the host cell’s chromosomes. • The plant cells containing the new DNA can be used to generate transgenic plants.

Figure 16.5 (c) Vectors for Carrying DNA into Cells

Getting New Genes into Cells • When a population of host cells is treated to introduce DNA, just a fraction actually incorporate and express it. • In addition, only a few vectors that move into cells actually contain the new DNA sequence. • Therefore, a method for selecting for transfected cells and screening for inserts is needed. • A commonly used approach to this problem is illustrated using E. coli as hosts, and a plasmid vector with genes for resistance to two antibiotics.

Figure 16.6 Marking Recombinant DNA by Inactivating a Gene

Getting New Genes into Cells • Other methods have since been developed for screening. • The gene for luciferase, the enzyme that makes fireflies glow in the dark, has been used as a reporter gene. • Green fluorescent protein, which is the product of a jellyfish gene, glows without any required substrate. • Cells with this gene in the plasmid grow on ampicillin and glow when exposed to ultraviolet light.

Sources of Genes for Cloning • Gene libraries contain fragments of DNA from an organism’s genome. • Restriction enzymes are used to break chromosomes into fragments, which are inserted into vectors and taken up by host cells.

Figure 16.7 Construction of a Gene Library

Sources of Genes for Cloning • Using plasmids for insertion of DNA, about one million separate fragments are required for the human genome library. • Phage l, which carries four times as much DNA as a plasmid, is used to hold these random fragments. • It takes about 250,000 different phage to ensure a copy of every sequence. • This number seems large, but just one growth plate can hold as many as 80,000 phage colonies.

Sources of Genes for Cloning • A smaller DNA library can be made from complementary DNA (cDNA). • Oligo dT primer is added to mRNA tissue where it hybridizes with the poly A tail of the mRNA molecule. • Reverse transcriptase, an enzyme that uses an RNA template to synthesize a DNA–RNA hybrid, is then added. • The resulting DNA is complementary to the RNA and is called cDNA. DNA polymerase can be used to synthesize a DNA strand that is complementary to the cDNA.

Figure 16.8 Synthesizing Complementary DNA

Sources of Genes for Cloning • If the amino acid sequence of a protein is known, it is possible to synthesize a DNA that can code for the protein. • Using the knowledge of the genetic code and known amino acid sequences, the most likely base sequence for the gene may be found. • Often sequences are added to this sequence to promote expression of the protein. • Human insulin has been manufactured using this approach.

Sources of Genes for Cloning • With synthetic DNA, mutations can be created and studied. • Additions, deletions, and base-pair substitutions can be manipulated and tracked. • The functional importance of certain amino acid sequences can be studied. • The signals that mark proteins for passage through the ER membrane were discovered by site-directed mutagenesis.

Some Additional Tools for DNA Manipulation • Homologous recombination is used to study the role of a gene at the level of the organism. • In a knockout experiment, a gene inside a cell is replaced with an inactivated gene to determine the inactivated gene’s effect. • This technique is important in determining the roles of genes during development.

Figure 16.9 Making a Knockout Mouse (Part 1)

Figure 16.9 Making a Knockout Mouse (Part 2)

Some Additional Tools for DNA Manipulation • The emerging science of genomics has to contend with two difficulties: • The large number of genes in eukaryotic genomes • The distinctive pattern of gene expression in different tissues at different times • To find these patterns, DNA sequences have to be arranged in an array on some solid support. • DNA chip technology provides these large arrays of sequences for hybridization.

Figure 16.10 DNA on a Chip

Some Additional Tools for DNA Manipulation • Analysis of cellular mRNA using DNA chips: • In a process called RT-PCR, cellular mRNA is isolated and incubated with reverse transcriptase (RT) to make complementary DNA (cDNA). The cDNA is amplified by PCR prior to hybridization. • The amplified cDNA is coupled to a fluorescent dye and then hybridized to the chip. • A scanner detects glowing spots on the array. The combinations of these spots differ with different types of cells or different physiological states.

Some Additional Tools for DNA Manipulation • DNA chip technology can be used to detect genetic variants and to diagnose human genetic diseases. • Instead of sequencing the entire gene, it is possible to make a chip with 20-nucleotide fragments including every possible mutant sequence. • Hybridizing that sequence with a person’s DNA may reveal whether any of the DNA hybridized to a mutant sequence on the chip.

Some Additional Tools for DNA Manipulation • Base-pairing rules can also be used to stop mRNA translation. • Antisense RNA is complementary to a sequence of mRNA. • The antisense RNA forms a double-stranded hybrid with an mRNA, which inhibits translation. • These hybrids are broken down rapidly in the cytoplasm, so translation does not occur. • In the laboratory, antisense RNA can be made and added to cells to block translation.

Figure 16.11 Using Antisense RNA and RNAi to Block Translation of mRNA

Some Additional Tools for DNA Manipulation • A related technique uses interference RNA (RNAi) which inhibits mRNA translation in the inactive X chromosome of mammals. • Scientists can synthesize a small interfering RNA (siRNA) to inhibit translation of any known gene.

Some Additional Tools for DNA Manipulation • The two-hybrid system allows scientists to test for protein interactions within a living cell. • A two-hybrid system uses a transcription factor that activates the transcription of an easily detectable reporter gene. • This transcription factor has two domains: one that binds to DNA at the promoter, and another that binds to the transcription complex to activate transcription. • An example is the yeast two-hybrid system.

Recombinant DNA and Biotechnology