Biotechnology The manipulation of organisms or their genes for –Basic biological research –Medical diagnostics –Medical treatment (gene therapy) –Pharmaceutical production –Forensics –Environmental clean up –Agricultural applications –Genetic components to make useful products

Humans have been manipulating the genetics of organisms for thousands of years

Animal husbandry/breeding

Biotechnology today Genetic engineering - the direct manipulation of genes for practical purposes –Manipulation of DNA is often called “Recombinant DNA” –Nucleotide sequences from two different sources are combined in vitro into the same DNA molecule

Recombinant DNA techniques usually start with DNA Cloning –Scientists clone pieces of DNA (or entire genes) in order to work with them in the laboratory –There are multiple methods to clone pieces of DNA or genes

DNA Cloning using bacteria and plasmids One common method to clone DNA involves using bacteria (often E. coli) Many bacteria contain an extachromosomal piece of DNA called a plasmid –Separate from main chromosome –Can replicate independently –Occur naturally in bacteria –Can be passed between bacteria

DNA Cloning using bacteria and plasmids Plasmids can be genetically engineered by inserting gene from another cell (plasmid is now recombinant DNA) Plasmids are used as cloning vectors – a piece of DNA used to carry foreign DNA into a host cell Plasmid is inserted back into bacteria by Transformation Bacteria is allowed to reproduce producing many identical bacteria with the plasmid (and desired gene)

Fig DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

Transformation Transformation was first performed in the laboratory by Griffith and later by Avery, MacLeod, and McCarty Bacteria can take up DNA only during the period a the end of logarithmic growth – cells are said to be competent (can accept DNA that is introduced from another source) E. coli are frequently used for transformation

E. coli competence can be induced under carefully controlled chemical growth conditions Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells E. coli cell membrane is weakened using ice cold CaCL 2 E. coli cells are then “heat shocked” to induce them to take up the plasmid Sterile technique must be used Transformation Lab – we will transform bacteria by introducing a plasmid that will convey resistance to the antibiotic, ampicillin Ampicillin kills bacteria by interfering with their ability to make cell walls

Biologists use Enzymes to “Cut and Paste” DNA Two important enzymes that biologists use for genetic engineering are restriction enzymes and DNA Ligase –restriction enzymes cut DNA molecules at specific DNA sequences –DNA Ligase stick lengths of DNA together –genetic engineers are able to use both enzymes to “cut and paste” DNA This is how plasmids are genetically engineered

Using Restriction Enzymes to Make Recombinant DNA Restriction enzymes come from bacteria In nature, bacteria use restriction enzymes for protection to cut foreign DNA (from invading viruses) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments

The most useful restriction enzymes cleave the DNA in a staggered manner to produce sticky ends Sticky ends can bond with complementary sticky ends of other fragments DNA ligase can close the sugar-phosphate backbones of DNA strands

Figure Restriction enzyme cuts the sugar-phosphate backbones. 3 5 DNA 3 5 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. DNA ligase seals the strands. Sticky end One possible combination Recombinant DNA molecule G C A A T T G GC CA TT A A TT A G GC CA TT A A TT A 123 Restriction site G GC CA TT A A TT A G G C C A T T A A T T A

DNA Fingerprinting Restriction Enzymes are also used for DNA fingerprinting (profiling) –Creating a pattern of DNA bands on a gel Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, a restriction enzyme will make many cuts Result: production of fragments of DNA of various lengths – Restriction Fragment Length Polymorphs (RFLPs) Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into different sized RFLPs

The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern Electrophoresis means “to carry with an electric current” Different sized RFLPs will be carried different distances by an electric current as they migrate through an agarose gel inside a gel box –Electricity is run through the gel box creating a positive end and a negative end Negatively charged DNA migrates from the negative end of the gel box through the pores in the gel to the positive end of the gel box Smaller RFLPs will migrate farther than larger pieces, spreading the RFLPs across the gel in a bar-like pattern Stain is used to make the DNA bands visible

Figure Mixture of DNA mol- ecules of different sizes Cathode Restriction fragments Anode Wells Gel Power source (a) Negatively charged DNA molecules will move toward the positive electrode. (b) Shorter molecules are impeded less than longer ones, so they move faster through the gel.

SEM photo of a 1% LE Agarose gel at 22kX magnification

Measuring fragment size Compare bands to known “standard” –Usually lambda phage cut with HindIII Nice range of size with a distinct pattern

DNA Fingerprinting – Uses Also called DNA Profiling Used to reveal a DNA pattern which is unique to an individual Crimework: rape and murder cases (forensics) Paternity suits Missing persons and unidentified bodies Immigration disputes Animal work - breeding

Polymerase Chain Reaction cloning a gene through genetic engineering can be time- consuming and requires an adequate DNA sample as starting material PCR technique allows researchers to amplify a tiny sample of DNA millions of times in a few hours DNA polymerase uses nucleotides and primers to replicate a DNA sequence in vitro, thereby producing two molecules Two strands of each molecule are then separated by heating and replicated again, so then there are four, double-stranded molecules After the next cycle of heating and replication there are eight molecules, and so on Number of molecules doubles with each cycle PCR is useful in amplifying tiny samples of DNA ranging from crime scenes to archaeological remains

Figure Cycle 1 yields 2 molecules Genomic DNA Denaturation Target sequence Primers New nucleotides Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Technique 123