The use of living things, biological systems and processes for the benefit of humans.

Tools for manipulating DNA 1. CUTTING DNA into fragments using restriction enzymes: these molecules cut at specific DNA sequences and are only found in prokaryotic organisms 2. PASTING Pasting DNA fragments together using enzymes called ligases. We can join fragments of DNA to make what is called “recombinant DNA”. DNA ligases found in many species including humans. 3. COPYING Making many copies of DNA (amplification) using DNA polymerases in the Polymerase Chain Reaction (PCR) technique. All organisms contain DNA polymerases as they all need to copy their DNA. 4. TRANSFERRING DNA into cells using vectors such as “plasmids”. This technique is called a transformation in prokaryotic cells.

1. Cutting DNA Restriction enzymes (molecular scissors) are found in prokaryotic organisms Cut DNA into smaller pieces (restriction fragments) in a controlled way – specific sequences of DNA called recognition sites.

Specificity Restriction enzymes are specific: The DNA and the enzyme need to be mixed together and incubated at a temperature that will result in maximum activity of the enzyme. Each restriction enzyme will only cut the DNA at a specific sequence of A, G, T and Cs. We call this place a recognition site.

Different restriction enzymes recognise specific recognition sites.

Cutting Specificity GAATTC CTTAAG GTT AAC GTTAAC CAA TTG CAA TTG When DNA is cut with a restriction enzyme the resulting fragments are left with either a short overhang of single stranded DNA called a sticky end or no overhanging DNA which is called a blunt end (snake demo) EcoRI – leaves sticky ends GAATTC CTTAAG HpaI – leaves blunt ends GTT AAC GTTAAC CAA TTG CAA TTG - site where enzymes cuts through the sugar phosphate backbone of the DNA strand.

What’s in a name! Restriction enzymes are named after the organism from which they were isolated. E.g. Escherichia coli EcoRI The Roman number indicates the order of discovery If another letter is placed in front of the Roman number it signifies a particular strain of the bacterium. R = resistance

Restriction Enzyme Restriction Site Overhang Type EcoRI E = genus Escherichia co = species coli R = strain RY13 I = first endonuclease isolated GAATTC CTTAAG STICKY BamHI B = genus Bacillus am = species amyloliquefaciens H = strain H GGATCC CCTAGG HindIII H = genus Haemophilus in = species influenzae d = strain Rd I = third endonuclease isolated AAGCTT TTCGAA HpaI pa = species parainfluenzae GTTAAC CAATTG BLUNT

Fragments are sorted by Gel Electrophoresis This technique is used to separate out fragments, obtained by a restriction digest, of DNA according to their size (length in base pairs). DNA fragments are separated into bands containing fragments of the same length by electrical separation in a gel matrix. DNA molecules migrate to the positive electrode, when an electric field is applied to the gel matrix, as they are negatively charged. This technique is used to isolate DNA fragments containing genes which are subsequently used to make recombinant DNA.

Fragments are sorted by Gel Electrophoresis

2. Pasting When two samples of DNA are combined using DNA ligases. Any 2 DNA strands can be joined that have complementary exposed nucleotides (i.e. cut with same restriction enzyme).

3. Copying (Amplification) of DNA using the Polymerase Chain Reaction The $300 million dollar man. “I was working for Cetus, making oligonucleotides (primers). They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. It was the first day of the rest of my life”. Kary Mullis 1972

Why PCR? To amplify a small amount of DNA into an analysable quantity E.g. crime scene, fossils etc

PCR Tools Taq DNA Polymerase – is an enzyme that works well at 72°C.

PCR Tools Primers: Synthetic short segments of DNA up to 25 nucleotides long. Probe for a specific sequence or gene along a strand of DNA. Hybridise with a sequence of bases on the template DNA through complementary base pairing. Indicate to Taq DNA polymerase where to start building the complementary strand by extending the primer.

Find the starting point for copying STR regions Select your primer Start region Sequence to be copied by extending the primer.

Thermocycling machine Step 1. Denaturation Step 1: Denaturing the DNA – 2 minutes T A C C G T A A A T G C C A T T Thermocycling machine Step 1: 92°C At this temperature the hydrogen bonds are broken resulting in two single strands of DNA.

Step 2. Attachment of Primers T A C C G T A A A T G T A A A T G C C A T T Step 2: 55°C Step 2: Primer annealing– 2 minutes The temperature is lowered to allow the primers to bind (anneal) to their complementary bases on each of the single strands of DNA.

Step 3: Extension Step 3: DNA synthesis – 1 minute T A C C G T A A A T G G C Taq A T T A T C Step 3: 72°C Taq G G T A A A T G C C A T T Step 3: DNA synthesis – 1 minute Taq DNA polymerase extends the DNA strand from the primers using the base pairing rule.

And you can repeat the three step cycle over and over!

PCR song http://www.youtube.com/watch?v=dD3faDLEvmY&feature=related

Fluorescent jellyfish 4. Transferring Jellyfish Fluorescent Because DNA is the same in all organisms, we should be able to take a piece of DNA from one organism and put it into another organism. You can change the way an organism looks or behaves! This process of taking DNA from one organism and putting into another is called transformation. Plasmid Jellyfish and plasmid DNA is cut with the same restriction enzyme.

Vectors Gene inserted into a vector that will carry the gene into the desired organism. Common vectors are: Viral vectors (eg. Adenovirus and retorovirus) – must have disease symptom genes removed first! Liposome vectors – small circular molecules surrounded by phospholipid bilayer Plasmid vectors – small circular piece of bacterial DNA. Plasmids are used as vectors in bacterial transformations.

Plasmids are not naturally attracted to bacteria! Bacterium

Transformation of Bacteria with a Recombinant DNA Plasmid Making the bacteria more ‘attractive’ to plasmids Plasmids are now attracted to the bacteria Bacterium CaCl2 solution

The Transformation Now give the bacteria some food and the right temperature to reproduce. Any bacteria with the plasmid inside will start making the jelly protein, that results in fluorescence. HEAT SHOCK

Gene Sequencing Gene sequencing is identifying the nucleotide order in a segment of RNA or DNA. A G G A C T C A T G G A G A A G A A C T T T . . . Our genome has been sequenced. We have 3,100,000,000 base pairs, what a big book!

Gene Cloning Making identical copies of sequences of DNA that code for proteins using plasmids extract plasmid from bacteria Cut plasmid DNA and DNA of the gene to be inserted with same restriction enzyme Paste 2 pieces of DNA using DNA ligase to create a recombinant plasmid. Add recombinant plasmid to bacterial culture, where some are taken up and replicate (called transformation) Isolate and analyse bacteria containing recombinant plasmids. PRACTICAL APPLICATION: Production of human growth hormone

DNA Profiling Compares base sequence of 2 or more individuals Short tandem repeats (STRs) and variable nucleotide tandem repeats (VNTRs): non-coding sections of DNA repeated many times between genes E.g. GAGAGAGAGAGAGA There are more than 10,000 STR loci in one set of human chromosomes!

DNA Profiling The repeat is present in all members of the population, but the number of repeats varies among individuals and is inherited. DNA profiling allows us to view these patterns in our DNA. Uses PCR and gel electrophoresis – smaller fragments will migrate further on the gel.

DNA Profiling

DNA Profiling Loci of STR regions found to vary from person to person with a high frequency 13 are used in America, but only 9 are used in Australia – why?