Title: The use of living things, biological systems and processes for the benefit of humans.
1- The use of living things, biological systems and
processes for the benefit of humans.
21. Manipulating DNA
- EL To learn how to cut and paste DNA
3Tools 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.
41. Cutting DNA
- Restriction enzymes (molecular scissors) are
found in prokaryotic organisms
5Specificity
- 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.
6Different restriction enzymes recognise specific
recognition sites.
7Cutting 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
GTT AAC
GTTAAC
CAA TTG
CAA TTG
- site where enzymes cuts through the
sugar phosphate backbone of the DNA strand.
8Whats 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
9Restriction 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 I first endonuclease isolated GGATCC CCTAGG STICKY
HindIII H genus Haemophilus in species influenzae d strain Rd I third endonuclease isolated AAGCTT TTCGAA STICKY
HpaI H genus Haemophilus pa species parainfluenzae I first endonuclease isolated GTTAAC CAATTG BLUNT
10Fragments 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.
11Fragments are sorted by Gel Electrophoresis
122. 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).
13Activities
- Watch DNAi animations
- Complete activity 9.1 Gel electrophoresis
analysis 9.2 Detecting traits in families.
14Reflection
- 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
152. Manipulating DNA
- EL To learn how to copy and transfer DNA.
163. 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
17Why PCR?
- To amplify a small amount of DNA into an
analysable quantity - E.g. crime scene, fossils etc
18PCR Tools
- Taq DNA Polymerase is an enzyme that works well
at 72C.
19PCR 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.
20Find the starting point for copying STR regions
- Select your primer
- Start region Sequence to be copied by extending
the primer.
21Thermocycling 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
Step 1 92C
- At this temperature the hydrogen bonds are broken
resulting in two single strands of DNA.
22Step 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 55C
- 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.
23Step 3 Extension
T A C C G T A A
A T G G C
Step 3 72C
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.
24And you can repeat the three step cycle over and
over!
25PCR song
- http//www.youtube.com/watch?vdD3faDLEvmYfeature
related
264. Transferring
Fluorescent jellyfish
- 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.
27Vectors
- 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.
28Plasmids are not naturally attracted to bacteria!
Bacterium
29Transformation of Bacteria with a Recombinant DNA
Plasmid
- Making the bacteria more attractive to plasmids
- Plasmids are
- now attracted
- to the bacteria
Bacterium
CaCl2 solution
30The 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
31Activity
- 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
32Reflection
- 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
333. Applications of DNA manipulation
- EL To explore the uses of DNA manipulation.
34Gene 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!
35Gene 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
36DNA 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!
37DNA 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.
38DNA Profiling
39DNA 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?
40Activities
- 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),
2526 (pg 457) - Ch 12 ch review qu 9, 10, 11 (pg 461)
41Reflection
- 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