Biotechnology -- Chap. 16.The use of biological systems for the

production of materials (most work is in the field of Genetic Engineering)

• process of altering biological systems by the purposeful manipulation of DNA – introduce specific foreign pieces of DNA into

host cells (often bacteria or viruses) which is then replicated by the host cells

– as the cell divides, a clone (exact copy) is produced

– cells containing the new DNA can be grown in any quantity and therefore, so is the protein transcribed by that DNA.

Genetic engineering

• The hard part is to “convince” the host cell to accept the foreign DNA– this is done by attaching the foreign DNA to a

carrier DNA molecule called a vector. – vectors are often bacteria’s plasmid (once

together they are now recombinant DNA) • Plasmids are molecules of DNA that are found in

bacteria separate from the bacterial chromosome. They: • are small, carrying one or a few genes• are circular • self-replicating

• This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.

The Process

• enzymes that cut the phosphate-backbones of DNA at specific base sequences (normally 4-6 bases) called restriction sites.

• exist naturally in bacteria in order to cut apart invading viral DNA

• cut parts of DNA are called restriction fragments

Restriction enzymes (also called restriction endonucleases)

• naming: ex) EcoRI– 1st letter is for genus - (Escherichia)– 2nd letter/s is for species - (coli)– 3rd letter is for the strain of the organism (R)– Roman numerals at the end typically

indicates the order of discovery (I)

Recombinant DNA video

Splicing and Cloning DNA Molecules (Fig. 16.2 in text)

• a restriction fragment of DNA can be duplicated by inserting it in a vector DNA molecule

• most common vectors are plasmids in bacteria and DNA viruses

• first, the vector and foreign DNA are cleaved by the same restriction enzyme in order for the sticky ends of both to match and then mixed together

• The enzyme ligase is added to join the vector and foreign DNA together making the foreign DNA an integral part of the plasmid

• This new plasmid is then taken up by other bacteria cells by a process called transformation

• Identification: Often the foreign DNA that was just inserted has other segments of DNA that identify it, such as resistance to antibiotics and the ability to produce color.

Steps in Cloning a Gene

DNA Sequencing

• the determination of the precise sequence of nucleotides in a sample of DNA.

• dideoxynucleotides are synthetic nucleotides (4 types, ddATP, ddGTP, ddCTP, ddTTP) that lack the -OH at the 3′ carbon atom and are each labeled with a "tag" that fluoresces a different color

• chain elongation proceeds normally until by chance, DNA polymerase inserts a dideoxynucleotide instead of the normal deoxynucleotide

• Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence