Gene Technology: DNA Manipulation and Sequencing

Gene Technology Overview

• Gene technology involves techniques for manipulating and sequencing DNA.
• This chapter covers recombinant DNA technology, PCR, cDNA cloning, gene libraries, gel electrophoresis, and applications of DNA technology.

Recombinant DNA Technology

• Recombinant DNA contains DNA molecules made in vitro with segments sourced from different origins.
• Gene cloning produces multiple copies of a gene of interest.
• The two primary goals include amplifying gene or producing protein products.

Tools Used in Recombinant DNA Technology

• Target DNA contains the gene of interest and can be extracted from organisms like human, animal, plant, fungi, and bacterial cells.
• Cloning vectors carry the gene of interest into host cells; bacterial plasmids are a common example of cloning vectors.
• Bacterial plasmids replicate separately from bacterial chromosomes and can be isolated from bacteria such as E.coli.
• Have selectable markers and unique restriction enzyme cleavage sites to indicate the insertion of recombinant DNA.
• Restriction enzymes, also known as restriction endonucleases, cut DNA molecules at specific places naturally in bacterial, with names derived from the source bacteria.
• Restriction enzymes recognize palindromic sequences, sequences identical when read in opposite directions on complementary strands, and makes a cut.
• Palindromic sequences contain 4-8 nucleotides
• Host cells are cells that accept recombinant DNA for cloning, such as E.coli.
• Can allow replication of recombinant DNA, maintain its structure, accept recombinant DNA via transformation and multiply the gene product.
• DNA ligase catalyzes the joining of DNA from two different sources, where the phosphodiester bond forms between sugar and nucleotides.
• Marker genes detect or "mark" the presence of certain gene sequence in the genome, enabling selection of transformed containing recombinant DNA.
• Examples of marker genes include antibiotic resistance genes (ampR, tetR), lacZ gene (encodes beta-galactosidase), and green fluorescent protein (GFP).

Steps of Gene Cloning

• Isolation of bacterial plasmid and the target gene.
• Insertion of target gene into a cloning vector.
• Introduction of recombinant DNA into a host cell via transformation.
• Amplification of the target gene by host cells.
• Selection and screening of recombinant clones.

Gene Cloning Steps with Bacterial Plasmids

• Plasmids are extracted, gene of interest from different sources, using the same restriction enzymes.
• Restriction enzymes cut the sugar-phosphate backbones of DNA strands in a staggered manner.
• Target gene fragments and plasmids get mixed
• In the presence of DNA ligase, base pairing between the plasmids and target genes occurs. This produces recombinant DNA.
• The enzyme DNA ligase facilitates the joining of DNA, producing stable recombinant DNA
• Recombinant DNA gets transferred into a host cell via chemical transformation using calcium chloride.
• Calcium chloride causes plasmids to stick to the bacterial cell wall.
• Heat shock is performed at 37-42 degrees Celcius for 45 seconds, changing fluidity of bacterial membrane and allowing plasmid to enter host cells
• Host cells get cultured in a ampicillin medium.
• Host cells with recombinant DNA reproduce through repeated cell division to form a clone of cells.
• selection involves identifying host cells that receive recombinant plasmid and marker genes.
• Successfully transformed cells will form colonies, where untransformed host cell colonies are not resistant to ampicillin.

Example: Insulin Production

• Example shown is the production of insulin using gene cloning
• Advantages of using gene cloning for insulin production include the product of insulin from human genes without triggering allergic reactions, while producing large amounts in short time.

Polymerase Chain Reaction(PCR)

• The polymerase chain reaction technique amplifies specific segments of DNA in vitro using replication of DNA in a test tube.
• PCR amplifies a small amount of DNA in less time.
• The amplified fragments are placed into a cloning vector, or used for other purposes.

Tools Required in PCR

• DNA Sample
• PCR Primers
• Taq Polymerase
• Free nucleotides (dNTPs)
• Thermal cycler/PCR/DNA Amplifier machine.

PCR Primers

• short single-stranded DNA sequences of around 20 nucleotide base pairs
• two primers used, one for each template.
• Taq polymerase synthesizes new DNA strands and is isolated from Thermus aquaticus which lives in hot springs.
• Taq polymerase is heat-stable and functions at temperatures up to 95°C.

Steps of the PCR Cycle

• Original DNA gets exposed to a high temperature to break hydrogen bonds between the two DNA strands via denaturation at 94-98°C.
• Fragment is cooled to allow primers to bind to each stand (annealing at 50-68°C)
• Free DNA nucleotides that are complementary with original strands added by taq polymerase to raise the temperature to form new DNA strands at 72°C.
• Each cycle produces identical DNA molecules.
• After 3 cycles, two molecules match te target seuqnce
• After 30 cycles, over 1 billion molecules over the target sequence.

Complementary DNA (cDNA) Cloning

• cDNA clones synthesized without the use of introns, using mRNA as a template for the first strand.
• Eukaryotic genes are large, preventing correct gene expression by bacterial hosts, but using cDNA overcomes this problem.
• Reverse transcriptase synthesizes the single strand of cDNA using mRNA as template.
• DNA polymerase is used to synthesize a second strand, for a double stranded cDNA molecule.
• cDNA clones get expressed in bacterial cells because of the introns and other segments of sequence.

Gene/DNA Library

• Gene/DNA library contains cloned DNA
• Two types: genomic library and complementary (cDNA) DNA library.
• Consists of DNA fragments that the entire genome of an organism.
• cDNA fragments that synthesize from mRNA will contain only coding regions of the DNA.

Gel Electrophoresis

• Cloned or amplified DNA fragments are separated based on size or mass.
• Gels are made of agrose or polyacrylamide and is submersed in a buffer solution that carries an electrical current.
• When current applied, the negatively charged DNA migrates toward the positive pole where larger frgments move slower.
• Visualized using fluorescent dyes.

Application of DNA Technology

• Recombinant DNA technology helps to examine fundamental questions about cells and new approaches to applied problems.
• Applications of DNA technology include criminal investigation, transgenic organisms, medicine, environment.

Application in Medicine

• identification of genetic mutation associated with genetic disease.
• gene therapy (introduction of gene into an afflicted individual for therapeutic puropses)
• Production of insulin for diabetic patients, genetically modified human growth hormones and produce vaccines for influenza A, hepatitis B and polio.

DNA Fingerprinting

• Analysis extracted from individuals
• Techniques rely on PCR amplification and gel electrophoresis
• DNA profile can be compared with millions of DNA profiles.
• DNA may come from blood, semen, bones, teeth, hair, saliva, urine or feces left at crime scene.

Transgenic Organisms

• Transgenic organisms: gentically modified oragnisms, where foreign genes have been incorporated.
• These organism produce specific, desirable and hertiable traits.

Applications in the Environment

• Transgenic microorganism like Pseudomonas bacteria break down hydrocarbons in oil and cleans up oil spillage.
• Transgenic microorganisms extract heavy metal in environments such as copper into copper sulfate or lead sulfate.
• Bioremediation uses using biological organisms to clean polluted areas such as saccharomyces cerevisiae.