MOLECULAR BIOLOGY NOTE
Genetic engineering = direct manipulation of the genome of an organism using biotechnology
Allows production of artificial drugs (eg. Insulin, hormones); artificial materials (eg.spider silk fibres
from recombinant silk produced in mammalian cells – thickness spider silk is stronger than silk);
artificial tissues (eg. Cartilages) and correction of genetic diseases (eg. Leukaemia has been treated
with immune cells that has been genetically engineered to wipe out cancer)
Recombinant DNA = combining two or more pieces of DNA that would not normally be found
together, using artificial means, rather than genetic recombination
Host vector systems
(=Weakened/ made safe)
Restriction enzymes
Used in bacterial defence mechanism – it can recognise
short sequences of double-stranded DNA (restriction
sites) as targets for cleavage leaving sticky ends or blunt
ends– allows bacteria to chop up potentially harmful
foreign DNA (eg. DNA from bacteria-infecting viruses)
Sticky ends = single-stranded overhangs which are
complementary to each other
Blunt ends = when cut straight down the middle of a
target sequence and leave no overhang (eg. Enzyme
called Sma I)
It’s found in bacteria and other prokaryotes
Restriction enzymes are named for prokaryote species they are isolated from (eg. Enzymes isolated
from E. Coli bacteria would begin with Eco as in EcoRI)
Isoschizomers = pairs of restriction enzymes specific to the same recognition
sequence but cuts in a different way/place
Compatible sticky ends Both cut at the
same genetic
sequence but by a
different enzyme
Thus one can H
bond with the
overhang
, Both cut by the same restriction
Plasmids
enzyme
➢ Found in bacteria
➢ Extrachromosomal (not part of bacterial chromosome), circular
DNA
➢ Often contain genes for resistance to antibiotics or production of
toxins
➢ Replicate independently of bacterial DNA and autonomously
(replicates themselves independently from the bacterial chromosome)
➢ Have been modified for use in DNA manipulation
➢ Polylinker/multiple cloning site = short DNA sequence containing two or more different
restriction sites for cleavage by restriction enzymes – it’s introduced into vectors to make
cloning easier by providing sites that allow cloning DNA, cut with any of a number of different
restriction enzymes into a single plasmid; allow a foreign piece of DNA to be inserted into that
region without disrupting the rest of the plasmid; standard feature of engineered plasmids for
making GMO; can be used to amplify the gene of interest by increasing the gene copy number in
the host; after bacterium replicates the gene of interest can be extracted out of the bacterium
➢ Exogenous = growing or originated from outside an organism
DNA ligase
DNA joining enzyme – if two pieces of DNA have
matching ends, ligase can link them to form a single
unbroken molecule of DNA
Using ATP as an energy source, ligase catalyses a
reaction in which the phosphate group sticking off the 5’end of one DNA strand is linked to the
hydroxyl group sticking off the 3’ end of the other. This reaction produces an intact sugar-phosphate
backbone
Restriction enzymes and DNA ligase are often used to insert genes into plasmids during DNA cloning
Target gene is flanked with EcoRI recognition sites and a
plasmid contains a signle EcoRI site
Separately digest/cut the gene fragment and the plasmid
with EcoRI to form fragments with sticky ends
Take the gene fragment and the linearized (opened-up)
plasmid and combine them along with DNA ligase. The
sticky ends of the two fragments stick together by
complementary base pairing
Once they are joined by ligase, the fragments become a
single piece of unbroken DNA resulting a recombinant
plasmid
, Possible outcomes: the cut plasmid could
recircularise (close back up) without taking in the
gene or gene could go into the plasmid forwards
but also flipped backwards since it’s two EcoRI
sticky ends are identical. Thus multiple types of
products can be made at different frequencies.
To only collect the correct plasmids, we can
analyse the bacteria using another restriction
digest to see if contains the right insert in the right orientation.
Recombinant DNA
marker
Transformation = bacteria taking up foreign DNA; key step in DNA cloning which occurs after
restriction digest and ligation
We can use antibiotic selection (eg. Ampicillin-resistant) and DNA analysis methods to identify
bacteria that contain the plasmid we’re looking for.
After transformation, bacteria are selected on antibiotic plates.
1. Bacteria are mixed with DNA
2. Bacteria are given a heat shock, which causes some of them to take up a plasmid – heat
shock makes the bacterial membrane more permeable to DNA molecules by causing a
formation of pores in the bacterial membrane, through which the DNA molecules can pass.
, 3. Plasmids used in cloning contain an antibiotic resistance gene. Thus all of the bacteria are
place on an antibiotic plate to select for ones that took up a plasmid.
4. Bacteria without a plasmid would die. Each bacterium with a plasmid gives rise to a cluster
of identical transformed bacteria called a colony
5. Several colonies are check to identify one with the right plasmid (eg. By PCR and DNA
sequencing)
6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein
production
We must check all the colonies (arose from single plasmid) since it is not necessarily the case that all
the plasmid-containing colonies will have the same plasmid since the gene can be combined in the
wrong way or not taken up. Backwards gene cannot be expressed in the bacteria to make a protein
but there are some cases it doesn’t. Gene must be inserted next to the promoter in the right
direction relative to it. If wrong way, wrong strand of DNA would be transcribed, and no protein
would be made.
Recombinant DNA analysis – Gel Electrophoresis
Stains all insert themselves in between the bases
of the DNA
Eg. Radioactive probe can be used eg. 32P + X-ray
or Fluorescent dye + UV light and take photo
In the gel electrophoresis, you must apply a
vector on its own and insertions on its own at the
side to allow comparison of the location of
different bands.
Molecular weight marker – known fragment size
Recombinant DNA analysis – Southern Blot
Combines DNA fragmentation, gel electrophoresis
and probe hybridization to analyse specific DNA
sequences
1. Restriction digest – RE cuts the DNA at
specific site and the number of fragments can be
amplified by PCR
2. Gel electrophoresis – desired DNA
fragments is separated
Genetic engineering = direct manipulation of the genome of an organism using biotechnology
Allows production of artificial drugs (eg. Insulin, hormones); artificial materials (eg.spider silk fibres
from recombinant silk produced in mammalian cells – thickness spider silk is stronger than silk);
artificial tissues (eg. Cartilages) and correction of genetic diseases (eg. Leukaemia has been treated
with immune cells that has been genetically engineered to wipe out cancer)
Recombinant DNA = combining two or more pieces of DNA that would not normally be found
together, using artificial means, rather than genetic recombination
Host vector systems
(=Weakened/ made safe)
Restriction enzymes
Used in bacterial defence mechanism – it can recognise
short sequences of double-stranded DNA (restriction
sites) as targets for cleavage leaving sticky ends or blunt
ends– allows bacteria to chop up potentially harmful
foreign DNA (eg. DNA from bacteria-infecting viruses)
Sticky ends = single-stranded overhangs which are
complementary to each other
Blunt ends = when cut straight down the middle of a
target sequence and leave no overhang (eg. Enzyme
called Sma I)
It’s found in bacteria and other prokaryotes
Restriction enzymes are named for prokaryote species they are isolated from (eg. Enzymes isolated
from E. Coli bacteria would begin with Eco as in EcoRI)
Isoschizomers = pairs of restriction enzymes specific to the same recognition
sequence but cuts in a different way/place
Compatible sticky ends Both cut at the
same genetic
sequence but by a
different enzyme
Thus one can H
bond with the
overhang
, Both cut by the same restriction
Plasmids
enzyme
➢ Found in bacteria
➢ Extrachromosomal (not part of bacterial chromosome), circular
DNA
➢ Often contain genes for resistance to antibiotics or production of
toxins
➢ Replicate independently of bacterial DNA and autonomously
(replicates themselves independently from the bacterial chromosome)
➢ Have been modified for use in DNA manipulation
➢ Polylinker/multiple cloning site = short DNA sequence containing two or more different
restriction sites for cleavage by restriction enzymes – it’s introduced into vectors to make
cloning easier by providing sites that allow cloning DNA, cut with any of a number of different
restriction enzymes into a single plasmid; allow a foreign piece of DNA to be inserted into that
region without disrupting the rest of the plasmid; standard feature of engineered plasmids for
making GMO; can be used to amplify the gene of interest by increasing the gene copy number in
the host; after bacterium replicates the gene of interest can be extracted out of the bacterium
➢ Exogenous = growing or originated from outside an organism
DNA ligase
DNA joining enzyme – if two pieces of DNA have
matching ends, ligase can link them to form a single
unbroken molecule of DNA
Using ATP as an energy source, ligase catalyses a
reaction in which the phosphate group sticking off the 5’end of one DNA strand is linked to the
hydroxyl group sticking off the 3’ end of the other. This reaction produces an intact sugar-phosphate
backbone
Restriction enzymes and DNA ligase are often used to insert genes into plasmids during DNA cloning
Target gene is flanked with EcoRI recognition sites and a
plasmid contains a signle EcoRI site
Separately digest/cut the gene fragment and the plasmid
with EcoRI to form fragments with sticky ends
Take the gene fragment and the linearized (opened-up)
plasmid and combine them along with DNA ligase. The
sticky ends of the two fragments stick together by
complementary base pairing
Once they are joined by ligase, the fragments become a
single piece of unbroken DNA resulting a recombinant
plasmid
, Possible outcomes: the cut plasmid could
recircularise (close back up) without taking in the
gene or gene could go into the plasmid forwards
but also flipped backwards since it’s two EcoRI
sticky ends are identical. Thus multiple types of
products can be made at different frequencies.
To only collect the correct plasmids, we can
analyse the bacteria using another restriction
digest to see if contains the right insert in the right orientation.
Recombinant DNA
marker
Transformation = bacteria taking up foreign DNA; key step in DNA cloning which occurs after
restriction digest and ligation
We can use antibiotic selection (eg. Ampicillin-resistant) and DNA analysis methods to identify
bacteria that contain the plasmid we’re looking for.
After transformation, bacteria are selected on antibiotic plates.
1. Bacteria are mixed with DNA
2. Bacteria are given a heat shock, which causes some of them to take up a plasmid – heat
shock makes the bacterial membrane more permeable to DNA molecules by causing a
formation of pores in the bacterial membrane, through which the DNA molecules can pass.
, 3. Plasmids used in cloning contain an antibiotic resistance gene. Thus all of the bacteria are
place on an antibiotic plate to select for ones that took up a plasmid.
4. Bacteria without a plasmid would die. Each bacterium with a plasmid gives rise to a cluster
of identical transformed bacteria called a colony
5. Several colonies are check to identify one with the right plasmid (eg. By PCR and DNA
sequencing)
6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein
production
We must check all the colonies (arose from single plasmid) since it is not necessarily the case that all
the plasmid-containing colonies will have the same plasmid since the gene can be combined in the
wrong way or not taken up. Backwards gene cannot be expressed in the bacteria to make a protein
but there are some cases it doesn’t. Gene must be inserted next to the promoter in the right
direction relative to it. If wrong way, wrong strand of DNA would be transcribed, and no protein
would be made.
Recombinant DNA analysis – Gel Electrophoresis
Stains all insert themselves in between the bases
of the DNA
Eg. Radioactive probe can be used eg. 32P + X-ray
or Fluorescent dye + UV light and take photo
In the gel electrophoresis, you must apply a
vector on its own and insertions on its own at the
side to allow comparison of the location of
different bands.
Molecular weight marker – known fragment size
Recombinant DNA analysis – Southern Blot
Combines DNA fragmentation, gel electrophoresis
and probe hybridization to analyse specific DNA
sequences
1. Restriction digest – RE cuts the DNA at
specific site and the number of fragments can be
amplified by PCR
2. Gel electrophoresis – desired DNA
fragments is separated
