GJHeducation

CIE International A-level Biology

19.1 (a, b, e - i)
The principles of genetic technology
 Have you got all BASES covered?
E N E I E H N O L O Y

I N V O L V E S H E R N S F E R

O F F R M E N S O F D N

F R O M O N E O R N I S M

O R S P E I E S O N O H E R

O F O R M R E O M B I N N D N

The 1st letters of the four nitrogenous bases that are found in the structure of DNA
have been left out of the definition above. Start this lesson by adding the missing
letters to work out the topic of today’s lesson and an initial definition
 Genetic technology
involves the transfer of
fragments of DNA from
one organism or species
to another to form
RECOMBINANT DNA
 This is actually fairly important……
Based on the fact that the genetic
code and transcription and translation
mechanisms are universal, the
transferred gene can be translated
within cells of the recipient
(transgenic) organism
RECOMBINANT DNA TECHNOLOGY

# 1a n e s
o f g e
a c ti o n m
Ex t r rg a n is
a n o
fro m
 This might just prove useful……
Some of the proteins which are produced
through genetic technology are relatively
small polypeptide chains (e.g. insulin = 51
amino acids). This meant that scientists
struggled to identify and isolate the gene
on DNA (in the nucleus) making it easier to
identify and isolate the mRNA
 19.1 (a-i)
QUIZ R1

The 1st round will

challenge your Biology,
literacy and numeracy
skills as you have to
convert FROM
NUMBERS 2 LETTERS
FROM NUMBERS 2 LETTERS
There will be a number of editions of this round of
the competition so keep your eyes peeled for this
title and background. Each time, a series of
biological statements will appear and each one has
a number as their answer. Those numbers then
correspond to a letter of the alphabet (e.g. 6 = F).
Buzz in when you have converted FROM NUMBERS
2 LETTERS and if you state the correct term, you’ll
get 2 TEAM POINTS.

FROM NUMBERS 2 LETTERS

FROM NUMBERS 2 LETTERS
 Nucleotide bases in a DNA triplet

 Different nucleotide bases on DNA

c D N A
 (DNA triplets in the genetic code) - 50

 Nucleotide bases changed in a substitution mutation

FROM NUMBERS 2 LETTERS

This is actually fairly important……
The c in cDNA stands for
complementary and this
polynucleotide is formed from mRNA
during this 1st stage
 2 TO THINK ABOUT
You have 4 minutes to discuss with 1 person
The mRNA strand for insulin is isolated but in order for the insulin gene
This 1 stage involves the
st the process needs the actual template strand
on DNA to be extracted,
of DNA. This involves a reaction to convert mRNA to cDNA and is

conversion of mRNA to
catalysed by an enzyme.

By considering that the role of this enzyme is

complementary
to convert mRNA to cDNA and byDNA using your
prior knowledge of protein synthesis and how
to(cDNA)
name enzymes,using REVERSE
in your pairs, discuss what
would be an appropriate name for this specific
TRANSCRIPTASE
enzyme
 Understanding check
Reverse transcriptase converts mRNA into cDNA
By considering the structure of mRNA and therefore the
resulting structure of cDNA, answer the following:

1. Suggest why cDNA is not considered to be “true”

DNA and describe what will have to happen in the
next stage as a result (2 marks)

2. Name the enzyme which will be involved in this

next step (1 mark)
 As complementary DNA (cDNA) was
converted from mRNA, it is only single-
stranded and therefore will need to be
converted into a double-stranded
polynucleotide in the next stage.

This will involve DNA POLYMERASE

 19.1 (a-i)
QUIZ R2

It’s CATCHPHRASE
TIME!
And in the words of the
legend that is Roy
Walker, just “Say what
you SEE”!
 Just
SAY WHAT YOU SEE
to get this one word
Whole number that Abbreviation used to
cannot be made by represent the
multiplying other transcription factor
whole numbers which binds oestrogen

PRIMER
“A short length of DNA, about 20 – 30
nucleotides long, which is artificially synthesised”
A primer is an essential requirement at this stage
because the enzyme DNA polymerase cannot act
on single stranded DNA. The primer is artificially
synthesised to be complementary to a sequence
of nucleotide bases at the 3’ end of the DNA.
Once the primer and DNA nucleotides are added
to the medium containing the cDNA, they will
move in and line up opposite the complementary
bases on the cDNA strand. In this way, the primer
will act as a point of attachment for the DNA
polymerase to begin to assemble the nucleotides
to form the new strand, just like it does in
“natural” DNA replication
FROM NUMBERS 2 LETTERS
In this round, you should do exactly as you did
before with the first 4 clues and convert FROM
NUMBERS 2 LETTERS. However, work out the
numerical answer to the 5th clue but leave it as a
number.

So your final answer should be 4 letters followed

by a number

FROM NUMBERS 2 LETTERS

FROM NUMBERS 2 LETTERS
 Kingdoms of living organisms

 Domains of living organisms

E c o R 1
 (Carbon atoms in deoxyribose) X 3

 (Polynucleotide strands in cDNA) x 18

 Polynucleotide strands in mRNA

FROM NUMBERS 2 LETTERS

 In case you are interested……
EcoR1 is an example of a restriction enzyme.
These proteins are named according to the
bacterium that they are taken from and by the
order in which they were found. For example,
EcoR1 tells us that this restriction enzyme was
taken from E-coli and was the 1st enzyme to be
found in that bacteria
Restriction enzymes are proteins that are
able to make cuts in the sugar-phosphate
backbone of DNA. They are used by
bacteria and archaea (where they are
naturally found) for protection against
attack by phage viruses as the cuts
prevent the viruses from making copies
of themselves. A restriction enzyme will
make a cut along the DNA once it
recognises its specific restriction site or
restriction recognition site
 THINK BACK & FORTH!
You have 4 minutes to discuss with 1 person
Baby anna was born on 02 02 2020 and to get her to sleep, her mother
A palindrome is a word, phrase or
constantly repeated “Was it a car or a cat I saw”
sequence that reads the same forwards as
By focusing on the 3 things highlighted in red
backwards. The restriction site of a
above, discuss whether anything catches your
restriction enzyme is normally a
eye and discuss how this could be related to
restrictionpalindromic
enzymes andsequence
the sequence below
G C G A A T T C A

C G C T T A A G T
 This is actually fairly important……
Restriction enzymes will recognise the
palindromic sequence and make a cut
within its recognition site on both the
top and bottom strands. In these
cases, the cuts are staggered cuts
 Understanding check
Restriction enzymes, like Eco-R1, make staggered
cuts in the DNA backbone

Once a restriction enzyme has found its

recognition site and made the cut, suggest what
will be left at the end of the DNA fragment
(1 mark)

At the end of the DNA fragment, there are

exposed bases which are known as sticky ends
EcoR1 makes its cut between adenine and guanine
on the backbone and this staggered cut leaves
exposed bases called sticky ends

A A T T C G T

Sticky ends G C A
G G

C C T T A A
 This is actually fairly important……
Although the term “sticky ends” doesn’t sound
particularly technical, these sections of exposed
bases are particularly important for the overall
process of genetic technology. Later in this
lesson, you will learn how the sticky ends of the
desired gene will be complementary to the
sticky ends of a plasmid (which was taken from
a bacterial cell and cut) and this allows them to
join to form recombinant DNA.
 YOUR TASKS

Answer questions 1 – 5 which

challenge your current
understanding of the enzymes
involved in recombinant DNA
technology
 A short length of nucleotides, known as a primer, is synthesised
to be complementary to a short section of the single-stranded
DNA. Following attachment of the primer, the enzyme RNA
polymerase can attach and begin to move along the newly
formed strand in the 5’ to 3’ direction. This enzyme catalyses
reactions that join complementary bases by hydrogen bonds and
forms phosphodiester bonds between the primer and the 1st
nucleotide and then between adjacent nucleotides after that.
The energy for these reactions is supplied by the condensation
of the released phosphate groups from the phosphorylated
nucleotides. The sequence of bases on the new strand is the
same as the sequence of bases on mRNA, apart from any uracil
base is replaced with an adenine base
RNA polymerase DNA polymerase
condensation hydrolysis
adenine thymine
 The unknown piece of DNA contains 1
restriction site for Kpn1. The length of the
unknown DNA, in the direction of the
restriction site on the plasmid, is slightly
less than 1000bp

Three fragments were obtained

 This might just prove useful……
A potential drawback of restriction
enzymes is that the specificity of their
restriction site means they cannot be used
to cut a DNA fragment if that particular
sequence of between 4 – 8 base pairs
doesn’t occur on that fragment.
RECOMBINANT DNA TECHNOLOGY

# 1b si s o f
y nth e
ica l s
Ch e m e s
g e n
FROM NUMBERS 2 LETTERS
 (Amino acids in the standard genetic
code) - 1

s s
 (The genes affected in haemophilia
and red-green colour blindness have loci
on this chromosome pair) - 4

FROM NUMBERS 2 LETTERS

 2 TO THINK ABOUT
You have 4 minutes to discuss as a pair!
ss is the abbreviation which
Single-stranded DNA goes(ssDNA)
before DNA to represent
can the product
be chemically
The 2 ssDNA molecules
of an automated instrument called a gene machine
synthesised using a gene machine. If two of these
sequences are synthesised, they can be used to form
Start will have been
yourdouble-stranded
synthetic discussionsDNA by (dsDNA).
ensuring
that you both agree on the identity of
ssDNAsynthesised to be
and then predict what could be
Take a further 2 minutes to discuss what
complementary
potentially formed if 2 to each
of these
must be true about the two ssDNAs for it
synthetic molecules were to be
other
to be possible to form
brought together
dsDNA
An automated instrument called a gene machine
can be used to chemically synthesise ssDNA
molecules of any desired sequence which are up
to 100 nucleotides in length. ssDNAs that are
complementary can be synthesised and then
hybridised to each other to form synthetic
dsDNA which has sticky ends.
In an upcoming lesson you will learn that DNA
fragments can be inserted into plasmids which
carry the gene into a bacteria. These DNA
fragments can be the dsDNA that is formed from
the ssDNAs made in a gene machine
 YOUR TASKS

Answer the three questions

on the worksheet which
challenge you to apply your
new knowledge of gene
machines
 Reverse transcriptase
Synthetic ssDNA is produced by the gene
machine meaning that there is no need to
convert mRNA into cDNA

DNA polymerase
Two synthetic ssDNA molecules can be
synthesised to be complementary to each other
and then hybridised to form synthetic dsDNA
meaning that cDNA doesn’t need to be used to
form double-stranded DNA
A short sequence of DNA
can be chemically
synthesised and inserted
into a fragment to add a
convenient restriction site
where it otherwise did not
occur
 By synthesising the altered
sequence of nucleotide bases
that results from a specific gene
mutation and replacing natural
DNA in cloned DNA, any effects
on the primary structure of the
polypeptide chain can be
studied
 In case you are interested……
Another use of synthetic DNA is to
act as a probe in DNA sequencing
to identify base sequences of
possible interest
 19.1 (a-i)
QUIZ R3

Round 3 is called THE

BIG REVEAL. 1 by 1, the
letters of this key term
will appear. Simply buzz
in when you know what
it is!
THE BIG REVEAL….

A transgenic organism is one whose

genome has been altered by
G ____ the transfer
N ____
T ____
____ R A ____
____ N ____
S ____ E ____ I ____
C
of a gene or genes from another species

Will you be
the 1st to get
it?
 This might just prove useful……
The term transgenic refers to the
organism which has the gene
transferred into its genome but the
actual cell which receives the DNA
fragment is known as a transformed
host cell
 Understanding check
During the process of genetic technology, a DNA fragment can
be transferred into a host cell in order to amplify the DNA
fragments
State whether the amplification of DNA fragments within a
transformed host cell is an example of an in vivo method or
an in vitro method. Briefly explain your answer
(1 mark)
The culture of transformed host cells is
an in vivo method as the process takes
place inside a living organism
 This is actually fairly important……
Although the actual amplification of
the DNA fragments within the
transformed host cells is an in vivo
method, the methods involved prior
this in order to prepare the DNA
fragments and other structures are in
vitro
 YOUR TASK

One particular cell that will often become a

transformed host cell is a bacterial cell.

Challenge your recall of the structure of

these cells from topic 1.2, by labelling the
cell on your worksheet

If you are unable to begin the labelling of a bacterial cell, ask for a
worksheet labelled STARS which gives you some assistance
 cell membrane

plasmid DNA

nucleoid DNA

cell wall

pili

flagellum
capsule mesosome

ribosome cytoplasm
 19.1 (a-i)
QUIZ R4

Round 4 is another
CATCHPHRASE!
With Roy Walker’s words
ringing in your ears, just
“Say what you SEE”!
 Just
SAY WHAT YOU SEE
to get this one word
In Physics, a quantity that has
magnitude and a specific
direction
VECTOR
(In Biology) A molecule used as a vehicle to carry
foreign genetic material into another cell
 Plasmids are circular loops of DNA
that are found in some bacterial cells.
As the genetic code is universal,
these plasmids can be used to carry
DNA from one cell to another and in
doing so, they will act as a vector.
But before any of this can happen,
the plasmid has to be removed from
the bacterial cell…
 RECOMBINANT DNA TECHNOLOGY

#2 pa re
d p re
v e a n t a s
o
Rem smid to a c
e p la r
th c to
a ve

PREVIOUS STEP
The desirable gene was extracted from an organism or the gene was
chemically synthesised
 This might just prove useful……
The most commonly used vectors are E-coli plasmids.
These small, circular DNA molecules include three
functional regions:

• an origin of replication
• a drug-resistance gene
• a region where the DNA fragment can be inserted
without interfering with plasmid replication or
expression of the drug-resistance gene
 Understanding check

An enzyme is used to cut the plasmid once it has

been removed from the bacterial cell

Name this type of enzyme and briefly describe

where this protein will make the cut on the
plasmid (3 marks)
The restriction enzyme will cut along the plasmid’s
backbone between two specific nucleotides which
are found within its restriction (or recognition) site
 1 TO THINK ABOUT
You have 4 minutes to discuss with 1 person
Once
The the plasmid
plasmid has
is been
cut removed
with thefrom the
same
bacterium, it is cut with a restriction enzyme
restriction enzyme that was used
By recalling any other details
to cut the DNA fragment. This that you
know about the cuts made by restriction
means thatdiscuss
enzymes, the resulting sticky
which specific
endsenzyme
restriction of the will
plasmid
be usedare
to make
the cut in the plasmid and ensure
complementary to the sticky ends that
you are able to explain why this specific
of the DNA
enzyme wouldfragment
be chosen
 This is actually fairly important……
Once in the transformed cell, the
structural gene (of the DNA fragment)
can be transcribed and translated to
synthesise the specific polypeptide.
For this reason, a promoter and
terminator region need to be added
prior to any insertion of the fragment
into the vector
 Prior knowledge check (topic 6.2)
Choose the most appropriate terms to fill the gaps in this
passage which describes the role of these two additional
regions (4 marks)
upstream
The promoter region is added __________ of the structural
gene and this is theRNA polymerase
section of the transcription unit where the
____ ____________ can bind. This region may also contain
transcription
different parts factors
for the attachment of various ___________
__________ to activate the binding of the enzyme.
Downstream of the structural gene, the addition of a
transcription
terminator region ensures that once the polymerase reaches
this point, ___________ will cease and the mRNA can detach
 RECOMBINANT DNA TECHNOLOGY

#3 A
h e D N
r t t
Inse t into the
g m e n
fra e c to r
v

PREVIOUS STEP
The plasmid DNA was removed from a bacterial cell and cut using the
same restriction enzyme that cut the gene
 Prior knowledge check (topic 6.1)

By verbalising your answer to a partner, name the

enzyme whose role in DNA replication is to join the
Okazaki fragments together to form the lagging
strand (1 mark)
DNA ligase
Again by verbalising your answer to a partner,
name the bond which will form between the
Okazaki fragments (1 mark)
phosphodiester bonds
 This is actually fairly important……
So DNA ligase is the enzyme that
catalyses the reactions that form
phosphodiester bonds between the
sticky ends of the plasmid and the
DNA fragment. This is called ligation
and the resulting molecule is known as
a recombinant plasmid (plasmid +
fragment)
 RECOMBINANT DNA TECHNOLOGY

#4
e r th e
n s f i d
Tra t p la sm
b in a n l l
re co m o s t c e
t h e h
into

PREVIOUS STEP
The DNA fragment was inserted into the vector to form a recombinant
plasmid
 Understanding check
The bacterial cells which will act as the host need to be treated
to increase the likelihood of successful uptake of the
recombinant plasmid. One particular method involves heat
shock treatment that occurs in the presence of some ions.

State whether these ions will be anions or cations and

explain your answer with reference to the structure of DNA
(2 marks)

Cations (positively-charged ions) will be used as they are

able to surround the negatively-charged parts, namely
the phosphate, of the DNA
 Understanding check
Cations are present during heat shock treatment to
surround the negatively-charged parts of the DNA
By verbalising your answer to a partner, name
another negatively-charged structure which will be
surrounded by the cations during the transfer of the
recombinant plasmid into the host cell (1 mark)

The phospholipids of the cell

membrane of the host cell
 During heat shock treatment, the bacterial
cell is subjected to alternating periods of
cold (0°C) and hot (42°C) in the presence of
calcium chloride. The positive calcium ions
surround the negatively charged parts of the
DNA and the phospholipids in the cell
membrane and this reduces the repulsion
between the bacterial cell membrane and
the foreign DNA. The result is a more porous
membrane which is more likely to allow the
recombinant plasmid (vector) to enter
 In case you are interested……
Alternatives to heat shock treatment
include electroporation, electrofusion
and transfection. In this latter process,
the DNA is packaged into a
bacteriophage (a virus) which is then
able to transfect the cell and replicate
within
 A VERY HIGH fraction?

The transformation
Following heat shock treatment,
what fraction of bacterial cells will
efficiency is very low
take up a single recombinant
so next…
plasmid?

A VERY LOW fraction?

RECOMBINANT DNA TECHNOLOGY

#5 ll s
t he c e
e nti f y n u p
Id e ta k e
h h a v n t
wh ic b in a
r e c o m
th e i d
pla s m

PREVIOUS STEP
The recombinant plasmid was transferred into a cell to form a
transformed host cell
 19.1 (a-i)
QUIZ R5

These CATCHPHRASE
rounds just keep coming
and I don’t think you
need me to tell you
what to do….
 Just
SAY WHAT YOU SEE
to get these two words
Abbreviation Sequence of
_______, used for the nucleotide
release, transcription bases on DNA
recapture factor which which code for
method binds oestrogen the amino acid
sequence

MARKER GENE
A marker gene is used to determine if a
nucleic acid sequence has been
successfully inserted into an organism’s
DNA. This is a key component of
recombinant DNA technology as the
transformation efficiency could be that
low that only one in several million cells
takes up the recombinant plasmid.
There are two sub-types of marker
genes…
 19.1 (a-i)
QUIZ R6

The 6th and final

round is a slightly
amended version of
the BIG REVEAL and
is called “PROVE
THAT YOU’RE ABLE”
Both of the sub-types of marker genes have
names that end in –ABLE. These four letters
have already been added to the end of the
name on the next slide, but the letters that
come before are yet to be seen. 1 by 1,
these letters will appear and basically it is
the team who recognise the name 1st and
buzzes in with the correct answer that will
get the 2 TEAM POINTS on offer.

So it is time to PROVE THAT YOU’RE ABLE!

PROVE THAT YOU’RE ABLE!

S E L E C T A B L E
____ ____ ____ ____ ____ ____ ____ ____ ____ ____

Will you be
the first to
get it?
 2 TO THINK ABOUT
You have 4 minutes to discuss with 1 person
A selectable marker protects the organism from a selective
The selective agent are
agent that would normally kill it or prevent its growth.
antibiotics as these drugs will kill
By focusing on the host cell involved
all of the cells that do not
in this lesson, discuss which selective
contain the
agent will beforeign
used andDNA, leaving
discuss what
only
you those with the
would expect to bedesired
found ingene
the
culture medium at the end
(in the recombinant plasmid) of this
stage of the process
PROVE THAT YOU’RE ABLE!

A screenable marker causes cells containing the gene to look

different. An example is a green fluorescent protein which
S C R E E N A B L E
makes
____ the____
____ cells ____
glow ____
green____
under UV ____
____ light and
____then a
____
specialised microscope can be used to see the individual cells

Will you be
the first to
get it?
 1 TO THINK ABOUT
You have 2 minutes to discuss as a class
So 5 steps are completed and the transformed host cells
have been identified

Finish this lesson with

one final discussion to
determine what the 6 th

and final step will be

RECOMBINANT DNA TECHNOLOGY

#6 c e lls
d h o st
f or m e lic a t e
Trans wed to rep vel
e a llo h e n o
ar re ss t e in
xp r o t
and e that the p
e so es te d
gen e ha rv
can b

PREVIOUS STEP
Using marker genes, or alternative methods, the cells which were
transformed were identified
 Specification point 19.1 (i) states that students
should be able to explain, in outline, how
microarrays are used in the analysis of genomes
and in detecting mRNA in studies of gene
expression

Following the culmination of this lesson,

research into the use of microarrays and add
these details to your notes to complete your
overview of the process of genetic
technology