What do you know about biotechnology? Where can you use it for?
-easier to grow and control. -Gene is the sequence of DNA, nucleotide base pairs -> similar in bacterial cells.
Basic techniques used to study genetic materials -isolate DNA from whatever cells -amplify DNA in vivo (transform the isolated DNA into empty bacteria cells)
Lyse cell membrane with detergent -> ppt DNA with alcohol (b/c DNA is insoluble in alcohol -> form layers. -> obtain ppt ed DNA by gently twirl with glass rod -> attach to glass rod -> solvate DNA in solution (ex.water..) DNA is relatively stable* -> can be stored in freezer for very long time
Genes may occupy only a small portion of the extracted DNA (only ~1% is coding region, the rest being non-coding) How to obtain specific sequence of DNA for study? -using technique called Restriction enzyme digestion*
Bacterium doesnot have defensive system -> using restriction enzyme as their immune system Why not digest host DNA?(why selectively cut foreign DNA?) -host DNA is methykated by a modification enzyme(methylase) to protect it from restriction enzyme’s activity
What is the advantage of stickyends?
Forming phosphodiester bonds
How are restriction enzyme useful?
But what if the DNA is relatively small amount?
Griffith’s experiemnt, (heat denatures the protein and RNA but not DNA) DNA is the transforming material. You can actually transform isolated foreign DNA into bacteria cells and amplification by growing the cells Transformation: process of introducing foreign DNA into a bacterial cell
Primers are synthesized, single-stranded DNA, 20-30 nucleotides long
Annealing temp depends on primer sequence&length Primers can anneal to the template DNA by forming hydrogen bond between nucleotides pairs elongation
Do you get the copies of targeted gene after cycle 1? Does transcription stop at the end of cycle 1? What tell transcription to stop? –the end of the gene (polyA??, hair pin structure just before polyA) -> No; synthesize the entire gene until polyA
Biotechnology• Manipulation of biological organisms or their components for research and industrial purpose• Usually manipulate DNA itself
How to study individual gene?• To study the function of individual genes, molecular biologists will cut them out of a genome and place them into bacteria• Why study gene in bacteria?
Basic techniques1. DNA isolation2. Restriction enzyme digest3. DNA amplification • Transformation and growth – In vivo • Polymerase Chain Reaction (PCR) – In vitro4. Gel electrophoresis
DNA IsolationBefore DNA can be manipulated, it needs to be isolatedfrom the cells.1.Disrupt cell membranes with a detergent • Example of detergent: SDS, Tween-202.Precipitate DNA with ethanol3.Obtain precipitated DNA and storage
DNA isolation How do you get specific sequence from the entire genome?
Restriction Enzyme Digestion• DNA must be cut into smaller pieces before they can be used in other techniques.
Restriction Enzymes• Molecular scissors• Restriction Endonucleases: digestive enzymes that recognize specific DNA sequences (known as Recognition site) and cut at specific points
Restriction Site• Typically 4-8 bp in length• Double-stranded DNA• Always palindromic: 5’ G A A T T C 3’ 3’ C T T A A G 5’ EcoRI recognition site What does palindromic mean? Same sequence on complementary strand in opposite orientation
Why use these enzyme?• Restriction enzymes are naturally found in bacteria• Restriction enzymes act as “immune system” of bacteria • Protect bacteria against DNA from other organisms (ex. bacteriophage (bacterial virus)) • Recognize and cut phosphodiester bonds of foreign DNA, not its own genome -> making foreign DNA harmless for the cell
Restriction enzyme digestion• Restriction enzyme recognize a palindromic DNA sequence in double-stranded DNA and cleave both strands• Resulting Sticky End: a single-stranded overhangs • Sticky ends with 5’ overhang • Sticky ends with 3’ overhang • Blunt ends
EcoRI digestion 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’ 5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang
PstI Digestion 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang
blunt ends – enzyme digests to make straight endsSmaI 5’ C C C G G G 3’ 3’ G G G C C C 5’ 5’ C C C 3’ 5’ G G G 3’ 3’ G G G 5’ 3’ C C C 5’
DNA LigaseT4 DNA ligase – used to chemically join sticky ends of DNA together
Recombinant DNAComplementary sticky ends from different pieces of DNA can be joined together – recombinant DNA
Amplification of DNA (in vivo)Transformation & Growth What is transformation?• Treat bacteria to make cell walls permeable to uptake of foreign DNA• Transformed bacterial cell grow and divide to amplify DNA
Amplification of DNA (in vitro)PCR PCR = Polymerase Chain Reaction•Powerful technique to produce millions of copies ofspecific DNA. _________________________________________ _________________________________________
Successful PCR reaction1) Need something to replicate and someplace to start1) Need something to open DNA and unwind it • AND something to stabilize it once unwound1) Need something to provide the primer to initiate synthesis2) Need something to synthesize the new DNA
• What you need: • Template DNA • dNTPs – nucleotides (dATP, dTTP, dCTP, dGTP) • Two specific Primers: short pieces (20-30 nucleotides) of synthetic single-stranded DNA • First is complementary to one DNA strand at the beginning of the target region • Second is complementary to opposite DNA stand at the end of the targeted region • DNA polymerase –Taq polymerase
Successful PCR reaction1) Need something to replicate and someplace to start Template DNA You can chose (chromosome, plasmid, etc)1) Need something to open DNA and unwind it • AND something to stabilize it once unwound Heat1) Need something to provide the primer to initiate synthesis You can chose the primers, and therefore specify EXACTLY what you want to amplify2) Need something to synthesize the new DNA DNA polymerase (Must be heat stable) *Taq polymerase
Taq polymerase• Isolated from Thermophilus aquaticus bacterium• These bacteria live in hot springs and has heat stable enzyme that can withstand extreme temperatures
3 steps for each PCR cycle1. DNA strand denaturation (95 oC) • Separate double strand DNA • Each strand becomes template strand2. Primer annealing (50 – 65 oC) • Short DNA pieces bind to temperate strands3. DNA strand synthesis (72 oC) • Produce new DNA strands
Gel Electrophoresis• Separate DNA through a gel medium using an electric currentWhy can we move DNA with electricity?• DNA has a negative charge. Therefore it will move toward positive electrode
Gel MediumThe gel medium can be made from: 1. agarose - seaweed extract 2. polyacrylamide - artificial polymer• The type of gel used is dependent on how well separated the DNA pieces need to be.• Polyacrylamide has higher resolution than agarose.
Liquid solutions of the gel is poured into a mould andallowed to set and solidify.
Loading DyeDNA is colourless.How do you know that it gets into the gel?• Coloured dyes are mixed with DNA to track distance travelled – Loading Dye
positive negativeelectrode electrode agarose gel
Separation DNA by sizeThe gel provides resistance for DNA movement. Short DNA • moves through gel easily • travels further in a set amount of time Long DNA • requires more effort to move through gel • does not move as far in a set amount of time
Visualizing GelDNA is colourless.How do you see where the DNA is after the separation is complete?• DNA is stained with ethidium bromide• UV light box is used to see fluorescent DNA bands