Recombinant DNA Technology

Technique that allows DNA to be combined from different sources – DNA code universal – Foundation of genetic engineering

Review What are enzymes? What function do they serve? What makes up the DNA sequence? What is the purpose of the genetic code?

Bacterial plasmids Recombinant DNA plasmids Transformation of plasmids Antibiotic selection of recombinant DNA pGLO plasmid – GFP gene (green fluorescent protein) – Antibiotic (ampicillin) resistant gene Transformation Lab Highlights

Recombinant DNA Technology

Discovery of Restriction Enzymes Bacteriophages are viruses that infect bacteria – Insert their genetic material (DNA) into the DNA of bacteria – Bacterial cells reproduce virus genetic material

Discovery of Restriction Enzymes

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In the 1960s, microbiologists discovered some bacteria protected from bacteriophage infection – Because they can restrict phage replication Restricted growth of phages occurred because the bacteria contained enzymes that could cut viral DNA into small pieces and prevent replication These enzymes were called restriction enzymes Discovery of Restriction Enzymes

Restriction Enzymes Primarily found in bacteria Given names based on genus and species names of bacteria from which they are isolated Cut DNA by cleaving the phosphodiester bond (in sugar-phosphate backbone) that joins nucleotides in DNA strand Enzymes bind to, cut (digest) DNA within specific sequences of bases called recognition sequence or restriction site Typically recognize 4, 6, or 8 nucleotide sequences

Restriction Enzymes Restriction enzyme - EcoRI Named because it was discovered in - Escherichia coli

Restriction Enzymes Some restriction enzymes cut DNA to create DNA fragments with overhanging single-stranded ends called “sticky” or cohesive ends

Cohesive Ends

Restriction Enzymes Some enzymes generate fragments with non- overhanging or blunt ends

Restriction Fragments Each cut of DNA creates restriction fragments A long strand of DNA may be cut several times by a restriction enzyme to create several restriction fragments

Restriction Enzymes Enzymes that produce cohesive ends are often favored over blunt-end cutters because DNA fragments with cohesive ends can easily be joined together. DNA from any source, bacteria, humans, dogs, frogs, dinosaurs, and ancient human remains can be digested by a particular restriction enzyme as long as it has the restriction site for that enzyme!

Recombinant DNA History In the early 1970s, a group of scientists joined together DNA from E. coli chromosomes and DNA from a primate virus – They isolated the DNA from each organism and cut them into fragments with EcoRI – They added the E. coli and viral DNA fragments to a reaction tube and added DNA ligase – They created a hybrid of the two DNA types, recombinant DNA

NIH and RAC Scientists were concerned about: – with what might happen if recombinant bacteria were to leave the lab – if such bacteria could transfer their genes to other cells – survival in other organisms including humans National Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC) to evaluate the risks of recombinant DNA technology and establishing guidelines

Recombinant DNA Technology Plasmids are circular DNA found in bacteria and replicate independently Plasmids are considered extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosome Plasmids can be used as vectors, pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA

Plasmid Vectors – used to clones specific DNA

Transformation Transformation – process for inserting foreign DNA into bacteria – Treat bacterial cells with calcium chloride solutions – Add plasmid DNA to cells chilled on ice – Briefly heat the cell and DNA mixture, plasmid DNA will enter the bacterial cell – Once inside bacteria, plasmids replicate and express their genes

Selection Joining of DNA fragments and plasmids by transformation can be inefficient – Some plasmids will rejoin and recircularize and not bond with any foreign DNA – During transformation, many cells will not take up DNA Recombinant bacteria, those transformed with a recombinant plasmid, must be separated from nontransformed bacteria as well as cells with plasmids without foreign DNA This process is called selection because it is designed to identify (select for) recombinant bacteria and prevent the growth of nontransformed bacteria and bacteria without foreign DNA

Transformation/Selection Animations ace/labbench/lab6/concepts1.html ace/labbench/lab6/concepts1.html hill.com/sites/ /student_view0/ch apter13/animation_quiz_1.html hill.com/sites/ /student_view0/ch apter13/animation_quiz_1.html ransformation1.html ransformation1.html

Transformation/Selection Resources Academic : – – – enthaler/Transformation.htm enthaler/Transformation.htm Pre-lab Resources: – – %20101%20laboratory/bacterial%20transformation/bacteria.htm %20101%20laboratory/bacterial%20transformation/bacteria.htm

Polymerase Chain Reaction Technique for making copies or amplifying a specific sequence of DNA in a short period of time.

PCR (Polymerase Chain Reaction) 1. Target DNA to be amplified is added to a thin- walled tube and mixed with deoxyribonucleotides (dATP, dCTP, dGTP, dTTP), buffer and DNA polymerase. Primers are added to the mixture; they are complimentary to nucleotides on opposite ends of the target DNA. 2. Tube is placed in a thermal cycler (gene cycler). This will take the sample through a series of reactions called the PCR cycle.

Thermal Cycler A.Denaturation – tube is heated to ~94-96ºC separating DNA into single strands B.Hybridization (annealing) – tube is cooled to ~60-65ºC allowing primers to bond to ends of DNA C.Extension (elongation) – temperature raised slightly and DNA polymerase copies the DNA by binding to primer and making a new strand DNA polymerase used in this reaction is Taq DNA polymerase from Thermus aquaticcus. Bacteria is adapted to live in hot springs it has enzymes that can withstand high temperatures necessary for PCR.

PCR At the end of one cycle, amount of DNA has doubled. Researchers can repeat cycle (usually times) to amplify millions of copies of DNA

Uses of PCR Studying gene expression Detection of viral or bacterial infections Diagnosing genetic conditions Amplifying trace amounts found at crime scenes Amplifying trace amounts found in ancient DNA

Gel Electrophoresis

Used to separate and visualize DNA fragments based on size Agarose gel is a semisolid material with small pores through which DNA can travel; fragments are separated by size – smaller fragments can travel further through pores than larger fragments Gel Electrophoresis

1. To run a gel, it is submerged in buffer solution that will conduct electricity 2. DNA samples are loaded into small wells and an electric current is passed through the gel 3. The sugar-phosphate backbone renders DNA with a negative charge; therefore DNA will move to the positively charged end 4.Because migration distance is inversely proportional to the size of the DNA fragment, large DNA fragments migrate short distances and small fragments migrate faster 5.Dyes are added to monitor DNA migration Gel Electrophoresis

Gel Electrophoresis Animations t/gelelectrophoresis.html t/gelelectrophoresis.html Gel Electrophoresis

Restriction Enzymes DNA cutting enzymes – Cuts double stranded DNA – Two incisions to cut DNA at sugar-phosphate bond on each strand Very specific – Enzymes recognize short nucleotide sequences – Enzymes cut the DNA at specific points within DNA For example – if the restriction enzyme cuts between a C and G then 5'GGCC3' 3'CCGG5'

Restriction Enzymes Each restriction enzyme recognizes a specific restriction site, a short nucleotide sequence Some restriction enzymes cut straight across the DNA Some restriction enzymes cut at offset nucleotides For example - G A A T T C C T T A A G G A A T T C C T T A A G G AATC CTTAA G

Restriction Enzymes Sequences that are cut at offset points produce overhanging pieces of DNA called “sticky ends” G AATC CTTAA G Sticky end

Restriction Enzymes

Why are restriction fragments useful? Recombinant DNA Gene isolation Genome mapping Gel electrophoresis Genetic engineering Gene therapy