Biotechnology

Biology for technology

Biotechnology

 The use of an organism, or a component of an organism or other biological

system, to make a product or process.

 Do you know examples of these?

 Wine, beer and
cheese production

Antibiotic
Biotechnology
production

Genetically
modified organisms
 Artificial selection

Traditional techniques involving

Biotechnology
microorganisms

DNA technologies
 DNA Analyze, manipulate,
and cut and paste
Technology pieces of DNA
  Clone: make many copies of a DNA fragment of interest
 Sometimes it involves inserting a target gene into a circular DNA
DNA Cloning molecule called a plasmid.
 The plasmid can be replicated and expressed making a protein.
Polymerase  DNA manipulation technique
chain reaction  PCR reactions produce many copies of a target DNA sequence
starting from a piece of template DNA.
(PCR)
  Technique to visualize DNA
fragments.
Gel  It separates DNA fragments
based on their size.
electrophoresis
 The fragments are stained
with a dye.
DNA sequencing  Determining the sequence of nucleotide bases in a DNA molecule.
DNA Cloning
Molecular biology technique
  Process of making multiple, identical copies of a particular piece of
DNA.
DNA Cloning  Conduct experiments
 Build plasmids (DNA fragments)
 Use bacteria as a factory to make a protein
  Genetic material of all organisms on Earth.
 Divided up into functional units called genes

Why DNA?  In transcription, the DNA sequence of a gene is copied to make an

RNA molecule
 In translation, the sequence of the mRNA is decoded to specify
the amino acid sequence of a polypeptide.
  The one gene, one enzyme hypothesis is the idea that each gene
encodes a single enzyme.
Genes and  Proposed by Sir Archibald Garrod.
enzymes  Confirmed by Beadle and Tatum using genetic and biochemical
studies of the bread mold Neurospora.
Next hypothesis:
If a mutant grew on minimal medium
with amino acids (but not vitamins), it
must be unable to make one or more
amino acids.

If a mutant grew on the vitamin medium

but not the amino acid medium, it must
be unable to make one or more vitamins.
One gene – one enzyme update

 Some genes encode proteins that are not enzymes

 Some genes encode a subunit of a protein, not a whole protein
 Some genes don't encode polypeptides, but functional RNA Molecules
 Typical DNA cloning procedure:
 Gene or other DNA fragment inserted into a circular piece of DNA
(plasmid) to produce a molecule of recombinant DNA*
Back to DNA  Recombinant plasmid is introduced into bacteria.
 Bacteria carrying the plasmid are selected and grown up.
Cloning
 Bacteria replicate the plasmid and pass it on to their offspring.

*DNA assembled out of fragments from multiple sources.

Step 1
Cut and
paste
Each restriction enzyme recognizes one
or a few target sequences and cuts DNA
at or near those sequences.

Restriction
Many restriction enzymes make
staggered cuts
enzymes and
DNA ligase

DNA ligase links two pieces of DNA with

matching ends to form a single,
unbroken molecule of DNA.
Restriction enzymes
DNA Ligase
1 2

3 4
Step 2
Transform bacteria
Specially prepared bacteria are mixed
with DNA plasmids

Plasmids used in cloning contain an Bacterial

antibiotic resistance gene. transformation

Heat shock causes some of them to take

up a plasmid.
Specially prepared bacteria are mixed
with DNA plasmids

Plasmids used in cloning contain an Bacterial

antibiotic resistance gene. transformation

Heat shock causes some of them to take

up a plasmid.
 Heat shock makes the
bacterial membrane
more permeable.

It causes the formation of

Heat shock pores in the bacterial
membrane.

DNA molecules can pass

through these pores.
Step 3
Select bacteria
Final step
Protein production and purification
Purification
Sometimes bacteria are used as "plasmid factories"

Making lots of plasmid DNA

Plasmid DNA might be used in further DNA cloning

Plasmid
production
Various types of experiments

Deliver genes to tissues

DNA Analysis methods
WHY IS IT IMPORTANT TO ANALYZE DNA?
 Amplification (PCR)
DNA Analysis Electrophoresis

Sequencing
 Technique to make many copies
of a specific DNA region in vitro

PCR relies on a thermostable DNA

polymerase (Taq polymerase)
PCR
It requires DNA primers designed
for the DNA region of interest.

The reaction is repeatedly cycled

 DNA polymerase

New strands of DNA, using existing strands as

templates
Taq
Polymerase
Hight-temperature resistant polymerase

Isolated from Thermus aquaticus

PCR
Cycle
 Visualize result of
PCR

Electrophoresis
Separation of
DNA fragments
according to
their size
 DNA samples are loaded into wells
(indentations) at one end of a gel

Gel An electric current is applied to pull

electrophoresis them through the gel

Small fragments move through the

gel faster than large ones
Made out of a polysaccharide
called agarose

Agarose is heated in a buffer and

allowed to cool
Gel

The gel is a matrix of agarose molecules

held together by hydrogen bonds that
form tiny pores.
Gel electrophoresis

 At one end, the gel has pocket-like indentations called wells, where the DNA is
loaded.
 The gel is placed in a gel box filled with a salt-containing buffer solution that can
conduct current.
 One end of the box is hooked to a positive electrode, while the other end is
hooked to a negative electrode.
 The end of the gel with the wells is positioned towards the negative electrode.
Gel electrophoresis
 Gel is stained with a DNA-binding
dye

Visualization DNA fragments can be seen

of DNA as bands under UV light

Each band represents a group of

same-sized DNA fragments
DNA Sequencing
Determining the sequence of nucleotides
Process of determining the sequence of
nucleotides

DNA
Target DNA is copied many times
Sequencing

Chain terminator nucleotides mark the

ends of the fragments
 Break the DNA of the
genome into many smaller
pieces

Sanger
sequencing
Fragments aligned based
on overlapping portions
to assemble the
sequences of larger
regions of DNA
 DNA Template

DNA polymerase enzyme

Sanger Primer
sequencing
DNA nucleotides

Marked dideoxy DNA nucleotides (chain terminating)

 Cycles: heating and cooling

Nucleotides bind
Sanger
sequencing
Eventually, ddNTPs bind and stop the chain

Capillary gel electrophoresis

Parallel sequencing

Next
Microscale generation
sequencing

Fast and low-cost