Download Biotechnology toolkit part 1 File

PART 1
CONTENTS
Introduction
Uses of Gene Technology
1. DNA extraction
2. Restriction Enzymes
3. DNA ligase
4. Reverse Transcription
5. Gel Electrophoresis
6. DNA Probes
7. PCR Polymerase Chain Reaction
8. DNA Sequencing
9.
Gene Cloning
10. Plasmids
11. Bacteriophages
12. Transgenesis
13. Tool Kit Vocab
Introduction
All genetic changes result from
* production of random variation
* selection pressures
Traditional breeding methods selectively choose desirable qualities. By selective breeding
the genetic make up of many species has been progressively altered under domestication.
These traditional methods of plant and animal breeding had three major limitations.



The production of better varieties was largely a matter of luck, since
recombination and mutation are both random processes.
Gene pools of different species are normally isolated from each other, so
geneticists were limited to crossing varieties of the same species, or in some
cases, closely related species.
It takes much longer to produce new varieties by selective breeding.
Molecular genetics has changed all that. Ever since it was discovered that the genetic
code is universal, the transplantation of genes between unrelated organisms has been a
possibility, and within the last 2 decades a reality.
Genes
Genes have two main components:

The protein coding region- contains the nucleotide triplet codes which code for
specific amino acids and the order they are arranged in. This is a universal code so
the same protein can in theory be made by any organism.

The promoter region- this region controls the gene expression. It regulates in which
tissue the gene should be expressed, at what time, and in response to what stimulus
the gene is transcribed. These promoters will only work within kingdoms and are not
as universal as genes. The promoter and the gene are usually physically separated.
USES OF GENE TEHNOLOGY
1. DNA Extraction
DNA must be extracted from the blood sample, piece of tissue, bacteria and virus cells.
DNA extraction must remove all the unwanted cell debris, membranes and proteins.
To isolate your own human DNA...
* To a 1/2 cup of water dissolve about 1/2 teaspoon of salt and add a squirt of dishwashing detergent. Save this for step 3.
* Swirl about 25 mls of water around your mouth for 30 seconds. This removes some
cheek cells. Spit into a disposable cup.
* Add about 2cm of the fluid to a test-tube (or a Fuji film canister) and add about 1cm of
the saline/detergent solution. Invert gently 3 or 4 times to mix well (but you don't want a lot
of froth). This will break open the many cheek cells you spat out, releasing the DNA
message that each cell must carry.
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* Layer on top some ice-cold ethanol (or methanol). Strands of DNA will be seen where
the two layers meet. Hook out the strands of DNA that form with a glass hook (or one
made from a plastic twist-tie) by slowly dipping up and down through the two layers.
To isolate plant DNA
* Blend 50g of Cauliflower in 200 mL of water for 30 seconds to get single cells.
* Strain through muslin cloth. Pour 15 mL of liquid into a screw cap tube with 3 drops of
detergent and shake gently for 5 seconds.
* Carefully layer to top of tube with cold alcohol (or meths). Use a hooked glass rod to
spool out strands of DNA at the interface.
Taken From http://talk.csifiles.com/showflat.php/Cat/0/Number/146627/Main/145305/
How DNA Extraction works…
Step 1: isolate the DNA from the rest of the cell
This is done by mechanically breaking the cells open, then using detergents and enzymes
to break down the cell walls and membranes. The detergents also break down the nuclear
membrane releasing the DNA.
Step 2: remove the unwanted cell debris
This is done by filtering the extract or by centrifuging the mixture. The filtrate (or the pellet
at the bottom of the centrifuge tube) will contain the DNA we want and unwanted proteins.
Step 3: remove the unwanted proteins
This is done by adding a protease enzyme that destroys proteins.
Step 4: precipitate out the DNA
The DNA can now be precipitated out by pouring a layer of ice-cold ethanol over the
surface of the filtrate.
The collected DNA can be now be used in some of the other techniques we will look at.
2. Restriction Enzymes
Restriction enzymes are used by bacteria to defend themselves against invasion by
bacteriophages (viruses that attack bacteria). Restriction enzymes (RE’s) act as chemical
scissors and cut DNA at specific sites. The ends created by restriction enzymes are either
blunt or sticky.
Restriction enzymes are named according to the kind of bacterium from which they are
obtained. Each restriction enzyme always cut the DNA at the same place, at the same
bases sequence.
The two ways of making a cut.
1
A blunt end
In this the DNA is cut straight across.
2
A sticky end
In this the DNA is cut staggered. They are called palindromic cuts because both ends
have sequences which read the same in the 5’ to 3’ direction.
Sticky ends are most commonly used.
After the DNA has been cut using restriction enzymes
 The ends produced by either blunt or sticky restriction enzymes can be attached
to some other DNA that has been cut by the same restriction enzyme.
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
Fragments can be separated by gel electrophoresis for detailed analysis
3. DNA Ligase
DNA ligase acts as a molecular stapler, joining DNA fragments. Its normal role includes
the joining of Okasaki Fragments in DNA replication, and in DNA repair.
The sticky ends of DNA are joined weakly by hydrogen bonds, the DNA ligase catalyses
the formation of phosphodiester bonds. Phosphodiester bonds occur between the
phosphates and the sugars on the sides of the DNA ladder.
Create sticky-ended DNA
fragments from two DNA
sources using the same
restriction enzyme.
Mix the DNA from the two
sources and allow
complementary ends to form
complementary base pairs.
This process of forming a
loose and temporary join by
hydrogen bonding is called
annealing.
Add DNA ligase to form a
permanent link.
4. Reverse Transcription
Many viruses do not carry their genetic
material as DNA, and carry their genetic
material as RNA.
3. Reverse Transcription
Reverse transcriptase is an enzyme produced by retroviruses, which include tumour
viruses and HIV.
Their genetic material is RNA and this is replicated indirectly in the host cell via a DNA
template- the reverse of what normally happens. The viral RNA is used as a template to
make a complementary sequence of DNA (cDNA), which is then used to make more viral
DNA from messenger RNA.
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