The use of living things, biological systems and processes for the benefit of humans.
1. Manipulating DNA
EL: To learn how to cut and paste DNA
Tools for manipulating DNA1. 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
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• 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
- site where enzymes cuts through the sugar phosphate backbone of the DNA strand.
GTT AACGTTAAC
CAA TTGCAA TTG
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 Escherichiaco = species coliR = strain RY13I = first endonuclease isolated
GAATTCCTTAAG STICKY
BamHI B = genus Bacillusam = species amyloliquefaciensH = strain HI = first endonuclease isolated
GGATCCCCTAGG STICKY
HindIII H = genus Haemophilusin = species influenzaed = strain RdI = third endonuclease isolated
AAGCTTTTCGAA STICKY
HpaI H = genus Haemophiluspa = species parainfluenzaeI = first endonuclease isolated
GTTAACCAATTG 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).
Activities
• Watch DNAi animations
• Complete activity 9.1 “Gel electrophoresis analysis” & 9.2 “Detecting traits in families….”
Reflection
Summarise in your own words – restriction enzymes, cutting, pasting and gel electrophoresis
•What learning was new today?•What learning was revision or built on what I already know?•What did I find most challenging and what strategies will I put in place to help me?•What percentage of the class did I spend on task and how can I improve this if needed
2. Manipulating DNA
EL: To learn how to copy and transfer DNA.
3. Copying (Amplification) of DNA using the Polymerase Chain Reaction
“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
• The $300 million dollar man.
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
• At this temperature the hydrogen bonds are broken resulting in two single strands of DNA.
Step 1: Denaturing the DNA – 2 minutes
Step 1: 92°C
T A C C G T A AA T G C C A T T
Step 1. Denaturation
• Step 2: Primer annealing– 2 minutesThe temperature is lowered to allow the primers to bind
(anneal) to their complementary bases on each of the single strands of DNA.
Step 2: 55°C
Step 2. Attachment of PrimersT A C C G T A A
A T G C C A T T
A T G
T A A
• Step 3: DNA synthesis – 1 minuteTaq DNA polymerase extends the DNA strand from
the primers using the base pairing rule.
Step 3: 72°C
Step 3: ExtensionT A C C G T A A
A T G C C A T T
A T G G C
A T T
Taq
G G T A A
A T C
Taq
And
you
can
repe
at th
e th
ree
step
cyc
le o
ver a
nd
over
!
PCR song
• http://www.youtube.com/watch?v=dD3faDLEvmY&feature=related
4. Transferring• 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.
Jellyfish
Fluorescent
Plasmid
Jellyfish and plasmid DNA is cut with the same restriction enzyme.
Fluorescent jellyfish
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
Activity
• In pairs, complete Activity 12.2 “finding a gene”
• Quick check qu 1-3 (pg 426), 4-6 (pg 431)
• Ch 12 review qu 3, 4, 5, 6, 7, 8, 12
Reflection
Summarise in your own words – copying (include PCR) and transferring
•What learning was new today?•What learning was revision or built on what I already know?•What did I find most challenging and what strategies will I put in place to help me?•What percentage of the class did I spend on task and how can I improve this if needed
3. Applications of DNA manipulation
EL: To explore the uses of DNA manipulation.
Gene SequencingGene 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
1. extract plasmid from bacteria2. Cut plasmid DNA and DNA of the gene to be inserted with
same restriction enzyme3. Paste 2 pieces of DNA using DNA ligase to create a
recombinant plasmid.4. Add recombinant plasmid to bacterial culture, where some
are taken up and replicate (called transformation)5. 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?
Activities
• Genetic Engineering: A model (Biol: The Common Threads, pg 175)
• DNA fingerprinting (Biol: The Common Threads, pg 179)
• Quick check qu 7-10 (pg 433), 11-14 (pg 437), 15-17 (pg 443), 18-21 (pg 448), 22-24 (pg 455), 25&26 (pg 457)
• Ch 12 ch review qu 9, 10, 11 (pg 461)
Reflection
Summarise in your own words gene sequeencing, cloning and DNA profiling
•What learning was new today?•What learning was revision or built on what I already know?•What did I find most challenging and what strategies will I put in place to help me?•What percentage of the class did I spend on task and how can I improve this if needed