Chapter 20 DNA Technology and Genomics

Biotechnology is the manipulation of organisms or their components to make useful products. Recombinant DNA is the result of the combination, in vitro, from 2 different sources into the same DNA molecule. Genetic engineering is direct manipulation of genes for practical purposes.

Cloning DNA cloning is a process used to make multiple copies of a desired gene or DNA segment. Cloning can also be used to make a desired protein product. Bacterial plasmids are the easiest sources to clone.

Restriction endonucleases are used to cut both DNA strands at specific sites by recognizing a particular short DNA sequence. Most restriction sites are symmetrical where the sequence of nucleotides is the same on both strands when read in the 5’  3’ direction. The most useful, cleave double strand backbones in a staggered way  “sticky ends”

Identification Identification of the clones with the genes of interest can be accomplished by looking for the gene itself or for the protein product. Nucleic acid hybridization is a process that utilizes the complimentary base pairing to a synthesized nucleic acid probe.

Polymerase Chain Reaction DNA cloning is the best method for preparing large quantities of a particular gene or DNA sequence. PCR is a method for quickly amplifying scanty or impure target DNA for analysis. This method requires a heat resistant DNA polymerase, nucleotides and 2 short single stranded DNA that serve as primers.

Restriction Fragment Analysis Restriction enzymes can be used to fragment DNA and then the fragments can be analyzed based on fragment size and banding pattern using gel electrophoresis. This process is useful when comparing 2 different DNA samples. Can be used to identify specific DNA when comparing the banding patterns to that of a known.

Differences in restriction sites found on homologous chromosomes are referred to as  restriction fragment length polymorphisms RFLP’s (rif-lips). RFLP’s are found in the non-coding sections of the genome and can serve as a genetic marker for a particular location on the genome, as in linkage maps. RFLP’s can be detected through a Southern Blotting technique.

DNA sequencing