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 12.1 “Restriction enzymes”

Reflection

• 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

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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

• 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 197)

• DNA fingerprinting (Biol: The Common Threads, pg 220)

• 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

• 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