Introduction

        In the 1950's, just after Watson and Crick inferred the structure of DNA, the genetic material of the cell, biologists formulated an important biological hypothesis -- the central dogma. The central dogma states that the instructions for making proteins are transcribed from DNA to messenger RNA (mRNA), and the mRNA instruction are translated into a sequence of amino acids in a protein. Proteins are major components of all organisms, including plants, because they catalyze the chemical reactions of metabolism and act as structural proteins. Subsequently, 45 years of molecular biology research have supported the central dogma, and shown it to be essentially correct and biologically universal in all living things. This includes the genetic code used to specify the amino acids that correspond to specific nucleotide sequences. Although chromosome structure is quite different from one species to another, the genetic code is nearly universal in viruses, and all organisms ranging from bacteria, fungi, plants, and animals to humans. This universality of the genetic code and all of the transcription and translation machinery in a cell makes it possible to identify and reproduce in a test tube virtually any gene from any organism; a process called gene cloning. Cloned genes can subsequently be manipulated and exchanged between species using genetic engineering to produce organisms with modified genetic composition for various purposes.

        One of the easiest and most straight forward methods for obtaining a gene is to replicate the DNA of the desired gene using a technique called polymerase chain reaction (PCR).  This technique involves designing primers at each end of the sequence we desire to replicate, and then using heat to separate the DNA strands.  The primers are then permitted to anneal to the sites in the DNA to which they are complementary, and heat stable DNA polymerase is then used to replicate the DNA from these primers to the oposite end of the strand of DNA.  In so doing, two molecules are formed from each DNA molecule you started with.  By subsequently repeating this cycle many times, the amount of DNA is doubled in each cycle.  Ultimately,  enough DNA is replicated that it can be manipulated into a bacterial plasmid referred to as a cloning vector.  This vector with the inserted DNA sequence can then be replicated in bacterial cells to produce multiple copies "clones" of the Vector plus insert.  

        Genetic Engineering is typically easiest with prokaryotes, e.g. bacteria, because many prokaryotes carry genes on isolated pieces of DNA called plasmids that replicate independently of the chromosomal DNA of the cell. This non-chromosomal DNA is usually not essential for prokaryotes in their natural environment, but the genes carried on a plasmid can be expressed as efficiently as those located on a chromosome. This means we can extract plasmids, make modifications to genetic composition of the plasmid, and put such modified plasmids into other bacteria or cyanobacteria for replication. In fact genes from any source can be replicated on bacterial plasmids, and thus, bacterial transformation systems are routinely used to replicate (clone) genes for manipulation because of the ease of working with these systems.

        Our special project will involve the following steps: (click on the hyperlink for each step to obtain directions for execution of that step.)

Step 1. Obtain a gene sequence we wish to clone using the GenBank data base at the National Center for Biotechnology Information (NCBI). This step will be executed as a homework assignment in lecutre.

Step 2.  Design nucleotide primers for a polymerase chain reaction (PCR) that would allow the amplification of the desired gene in a test tube.This step will be executed as a homework assignment in lecture.

Step 3.  PCR amplification of a gene. Use the nucleotide primers for Polymerase Chain Reaction amplification of the specific sequence.

Step 4.  Identification and characterization of the DNA PCR product by electrophoresis.

Step 5.  Ligation of the PCR product into the cloning vector to create a plasmid carrying an inserted sequence.

Step 6.  Transformation of E.coli cells with this vector containing the inserted sequence and growth of a culture containing the cloning vector with the inserted sequence.

Step 7.  Extraction, purification, and analysis of the plasmid (cloning vector with inserted sequence) from the cells.  Including digestion with restriction enzymes to remove the plasmid insert..