Aim: What are some techniques used in DNA engineering?

Making cDNA Complimentary DNA (cDNA) is made in vitro. mRNA from the selected gene is isolated Reverse transcriptase creates a cDNA. mRNA is degraded The second strand of DNA is made.

Nucleic Acid Hybridization 1) Transfer cells to filter 2) Treat cells on filter to denature DNA 3) Add a cDNA radioactive probe; hybridization will occur 4) expose photographic film; exposed areas are colonies with the cloned DNA 5) compare to original petri dish.

PCR (polymerase chain reaction) (in vitro) This technique is used to make many copies (billions) of DNA fragments in a few hours.. It involves heating, cooling, & replication. The DNA is incubated in a test tube with special, heat-tolerant DNA polymerase, a supply of nucleotides, and short pieces of single- stranded DNA as a primer.

PCR (polymerase chain reaction) Devised in 1985, PCR has had a major impact on biological research and technology. PCR has amplified DNA from a variety of sources: fragments of ancient DNA from a 40,000-year-old frozen wooly mammoth, DNA from tiny amount of blood or semen found at the scenes of violent crimes, DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV.

Gel Electrophoresis Gel electrophoresis separates macromolecules - nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field For linear DNA molecules, separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel. After treating long DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis. The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.

Gel Electrophoresis In gel electrophoresis, the restriction fragments from two differnt alleles will produce different band patterns, allowing us to distinguish them apart.

Southern Blotting technique Southern blotting (Southern hybridization) allows us to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size. Bathing this sheet in a solution containing a radioactive probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest We can visualize bands containing the label with autoradiography.

Southern Blotting

Sanger Method This method is used to determine the nucleotide sequence of DNA. It is similar to PCR, however, special nucleotides, called dideoxynucleotides, are used to copy the DNA as tiny fragments. The order of these fragments via gel electrophoresis can be interpreted as the nucleotide sequence.