B.

APPLICATIONS SYLLABUS

8. ISOLATING, CLONING AND SEQUENCING DNA

(a) Discuss the natural function of restriction enzymes.

Restriction enzymes are part of the bacterial defence system

- break down viral DNA by hydrolysing the sugar phosphate backbone (phosphodiester bond)
- restrict the ability of viral DNA to infect the host bacterial cell
- Host DNA is protected through methylation (methyl groups added to C or A groups)

(b) Explain the formation of recombinant DNA.

Recombinant DNA: DNA from 2 or more different sources

Production of restriction fragments

- Restriction enzymes recognise specific palindromic nucleotide sequences in the DNA (restriction sites) E.g.
‘GAATTC’ for EcoRI
- Cleave both strands of DNA by hydrolysing phosphodiester bonds, producing one or more linear ds-DNA
fragments with either sticky or blunt ends
- DNA fragments with sticky ends short extensions that anneal (through the formation of H bonds) with
complementary extensions on other molecules that are cut with the same RE
- Blunt ends do not bind specifically to other blunt ends
require the adding of a single chain of nucleotides
to each end of the molecule with the aid of terminal transferase OR adding linkers (short pieces of DNA with a
restriction site)

Restriction fragments are then sealed using DNA ligase, which catalyses the formation of phosphodiester
(covalent) bonds between the restriction fragments

(c) Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid and describe the properties of
plasmids that allow them to be used as DNA cloning vectors.

Procedures for cloning a eukaryotic gene in a bacterial plasmid:

1. Isolating the required gene of interest

- Either through polynucleotide synthesizer (exact gene sequence must be known), digesting the
existing DNA molecule with RE, or synthesizing cDNA from mRNA using reverse transcriptase
2. Recombination
- Insert the gene of interest into a plasmid to form a recombinant DNA, to promote the uptake of the
gene by the host cell.
- Necessary because linear DNA fragment is unstable compared to circular DNA molecules in bacteria,
and it cannot replicate itself
-Method: DNA and vector cut separately with same RE to produce complementary sticky ends to
facilitate complementary base pairing, then combined together with addition of ATP and DNA ligase
3. Transformation
- Transformation: Process by which a host cell assimilates external DNA, resulting in genotypic and
phenotypic change
- Uptake of recombinant DNA by host bacterial cell so that it can replicate. Host cells possess the
protein synthesizing machinery for manufacturing the proteins coded for by the gene of interest
- Fast reproductive rate of bacteria ensures multiple copies of GOI and their products
- Method: Mixture of recombinant DNA and host cells subjected to CaCl2 and brief heat shock

makes holes appear briefly in cell surface membrane, increasing permeability for uptake of foreign
 DNA (become competent)
- Other methods: Electroporation, Liposome, Gene gun, Viral insertion
- Transformation not efficient
4. Screening for desired transformed cells
- Correctly identify the cells containing gene of interest
- Requires the use of selective medium and genetic markers on the vector, involves insertional
inactivation
-Method (i): Replica plating
nd

involves 2 antibiotic resistance genes on the vector, when gene was inserted, disrupted the 2
selectable marker. Successfully transformed cells will not survive and reproduce in the presence of
that antibiotic

Procedure - Gently press a velvet pad onto the original master plate (e.g. with ampicillin). Press the
pad onto a second plate containing the second selective agent (e.g. tetracycline) Only the
recombinants lose tetracycline resistance.
- Method (ii): Blue-white screening

involves 1 antibiotic resistance gene and the LacZ gene (codes for beta-galactosidase that catalyses
X-gal to blue product) on the vector, agar nutrient plate contains antibiotics, substrate X-gal and
inducer IPTG.

Insertion of DNA fragment inactivates LacZ gene, hence recombinant colonies appear white; those
with re-annealed plasmid appear blue
5. Large scale production
- Correct recombinant DNA further multiplied by transferring the selected bacterial colony into a
liquid nutrient medium

Properties of plasmids that allow them to be used as DNA cloning vectors

- Plasmids have origin of replication, enable host cell polymerase to replicate independently. Copies of
recombinant DNA molecules can thus be produced and passed on.
- Plasmids have high copy number per host cell.
- Large quantities of the recombinant DNA molecule can be obtained from each cell.
- Plasmids have selectable markers
Genes that confer readily selectable phenotypic traits (e.g. resistance to antibiotic)
Gene of interest is inserted in between one of the selectable markers (insertional inactivation)
Most contain at least two selectable markers – One intact to select transformant, and the other to
detect inactivation

Note: Insert DNA that are too large (>10kb) are not replicated efficiently as under selective pressure, the large
plasmids would tend to delete non-essential genes (i.e. foreign inserts)

(d) Explain how eukaryotic genes are cloned using E. Coli cells to produce eukaryotic proteins to avoid the
problems associated with introns.

Bacteria lack:
- Ability to perform post-transcriptional modifications
- Ability to perform post-translational modifications
- Ability to recognise eukaryote’s promoter

- mRNA extracted from eukaryote cells and used as a template for the synthesis of cDNA, using enzyme
reverse transcriptase (isolated from retroviruses).
- Introns will not be present
- Extra guanines added to each end of DNA strand to create sticky ends, extra cytosines added to ends of cut
plasmids (enable complementary base pairing)
(e) Distinguish between genomic and cDNA libraries

Gene or DNA library: a collection of DNA fragments derived from the genome of an organism, or some other
collection, and cloned randomly into suitable cloning vectors.
Purpose: Obtain sequence of genes, allow gene expression to obtain particular product, preserves genes of a
species that are in danger of extinction

Feature Genomic Library cDNA library

Starting material for Contains total cellular/genomic DNA
construction (coding + non-coding sequences)
Content Requires entire chromosomal
isolation
Source Can be obtained from any tissue
Contains total coding sequence
(exons) of a particular cell type,
expressed at a particular stage of
the development of the organism
Requires total mRNA isolation
Should be isolated from particular
tissue where protein is likely to be
expressed in large amounts

From plants and animals that have

Generally from prokaryotes, yeast and
fungi where complete genomic library
is not too large to be unmanageable
Process Cleaved with RE into small fragments
before ligating into cloning vector
Size Large genome size – too many
fragments to screen
Isolation of gene products in Unlikely due to the lack of post-
bacteria transcription machinery in bacteria
large genome size and introns that
can complicate expression of genes
Isolation of mRNA by hybridising
poly(A) tail with oligonucleotides of
poly(T) in a column matrix.
mRNA reverse-transcribed into
cDNA, forming RNA-cDNA hybrid.
RNA strands nicked with
ribonuclease, DNA polymerase used
to replace strand before ligating into
cloning vector
Abundance of mRNA in particular
tissue type – facilitates screening of
recombinant clones by increasing
chance of obtaining correct clone
because these tissues usually
contain more of these specific
sequences
Possible as the non-coding
sequences are absent. Expression of
gene need not involve post
transcription modifications

(f) Outline 2 important proteins and other products that can be produced by genetic engineering technique
(e.g. human growth hormone, anti-thrombin, etc.)

Insulin

Structure: Peptide hormone, 2 chains, secreted by β cells of Islets of Langerhans

Function: Regulate blood glucose levels (refer to Homeostasis core syllabus)
Problems: Deficiency leads to diabetes mellitus
Special Preparation: GOI is a cDNA molecule prepared from mRNA of insulin. DNA polymerase makes single-
stranded cDNA double stranded.

Human Growth Hormone (HGH)

Structure: Peptide hormone with 191 amino acids, secreted by anterior pituitary gland. During its production,
intermediate molecule has a signal peptide (26 aa) attached that is cut free during secretion.
Function: Stimulate growth by increasing cellular intake of amino acids, protein synthesis and use of fats as
body fuel.
Problems: Deficiency leads to dwarfism in children
Special Preparation: EcoRI used to remove signal sequence + genes for first 24 amino acids. Gene for the 24 aa
is synthesised separately and then attached to existing DNA fragment by DNA ligase.

Anti-Thrombin

Structure: Glycoprotein with 432 amino acids, secreted by liver

Function: Inactivates thrombin (+ve feedback on its own formation; catalyses fibrinogen
fibrin;
stabilises/strengthens fibrin by activating enzyme factor XIII to XIIIa which in turn catalyses formation of
covalent cross-linkages between the loose fibrin strands) and other clotting factors, prevents spread of clot by
rapidly inactivating clotting factors that are carried away from immediate site of the clot by the flowing blood.
Problems: Deficiency leads to venous thrombosis
Special Preparation: Human anti-thrombin gene isolated and attached to animals’ β-lactoglobulin gene
promoter, inserted into fertilised embryo of animal via microinjection. Transgenic embryo placed into
surrogate host which gives birth to transgenic animal, anti-thrombin secreted in milk of transgenic animal.

(g) Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this
procedure.

PCR used for amplification of a specific sequence of DNA in a short period of time.
X
Each PCR cycle results in doubling the number of DNA sequences replicated. X cycles will yield 2 strands of
target DNA.

Components for PCR:

Source DNA containing the segment to be amplified
Oligonucleotide primers
Synthetic ssDNA (20-30bp)
Initiate DNA synthesis
Complementary to sequence flanking target DNA segment (2, one to each strand of DNA
double helix)
Taq polymerase
Thermo stable
Stable at 95°C, optimal performance at 72°C
Deoxyribonucleoside triphosphates (dNTPs)
Substrates for DNA replication (dA/T/C/GTP)
Procedures of PCR:
- Denaturation: Brief heat treatment (up to 95°C) to separate DNA double helix by breaking H bonds.
Each strand as template for synthesis of complementary strand.
- Annealing: Cooling to 54-68°C in presence of forward and reverse DNA primers to allow attachment
(complementary base pairing). Primers provide free 3’OH end for DNA polymerase to work on.
- Elongation: Taq polymerase catalyses synthesis of complementary DNA strand at 72°C (optimal
temperature). Add free dTPs in a 5’
3’ direction

Advantages of PCR Limitations of PCR

Each round of PCR doubles number of target DNA.
Amount of desired sequence increases exponentially.
Thermocycler is easy to use, can clone large amounts
of DNA in a relatively short time.
Highly sensitive – target sequence can be amplified
Limited amount of DNA obtained due to strand
breaks or failure of DNA to dissociate from other
macromolecules during purification. Amount of
enzyme is finite, competition increases as number of
strands increases.
Requires prior knowledge of DNA sequence for
even when only a minute amount of DNA source is
available
Contributes in clinical (detecting genetic disease at
embryonic stage), forensic, archaeology and
palaeontology (amplified to have large amounts to
analyze)
Dependable – PCR will continue operating even
though conditions are constantly changing. Allows the
amplification of a broad range of nucleic acid (badly
degraded/difficult to be isolated)
synthesis of primers. Time is required for the design
and synthesis of primers flanking the target region.
Taq polymerase lacks 3’
5’ proofreading ability,
leading to high error rate due to mismatch of free
dNTPs. Final product a mixture of extremely similar,
but not identical DNA sequences.
Risk of contamination – introduction of non-target
DNA sequences might lead to a mixture of products
being amplified (if foreign DNA is complementary to
primer)
DNA fragment lengths are limited to 0.1 – 5 kb.
Further increase decreases efficiency as the
polymerase tends to ‘fall off’ the DNA before
replication is complete.

(h) Explain how gel electrophoresis is used to analyse nucleic acids and proteins and to distinguish between
two alleles of a gene.

Gel electrophoresis: Technique by which charged molecules are separated based on their size or charge, by
passing them through a gel (solid but porous matrix) within an electric field

Agarose Gel Electrophoresis is used for separation of DNA; SDS-PAGE (Sodium dodecyl sulphate
polyacrylamide gel electrophoresis) is used for analysis of proteins

Procedures for Agarose Gel Electrophoresis

- A slab of agarose gel is placed in buffer solution which allows conduction of electricity to
generate the electric field
- Gel pre-cast with little indentations (wells) at one end, using a comb. Each well corresponds to
one lane.
- DNA sample mixed with dense loading dye(helps DNA sink to bottom, and monitor progress of
electrophoresis)
- Markers (prepared mixtures of DNA fragments of known size) are run alongside, form basis of
comparison.
- When the current in turned on, -ve charged DNA fragments move towards anode.
- The meshwork of polymer fibers in agarose gel impedes movement, such that shorter DNA
fragments move faster than longer fragments due to less resistance. Rate of movement inversely
proportional to size of molecule
- DNA sample will separate to form discrete bands on the gel
- Ethidium bromide is used to visualize the bands, under UV light, as the dye bound to DNA
fluoresces.

Procedures for SDS-PAGE

- Performed in polyacrylamide gels.
- Similarly, gel pre-cast with little indentations (wells) at one end, using a comb. Each well
corresponds to one lane.
- Proteins first treated with SDS (anionic detergent), denaturing the proteins (breaking
hydrophobic interactions and coating polypeptide with many –ve charge, thereby linearising
them), thus proteins are separated by mass alone.
- Mixture of denatured proteins applied to gel, and apply current. Smaller proteins move faster
than larger proteins.
To distinguish between two alleles of a gene
- Alleles (alternative forms of a gene) are expressed differently due to variation in sequence of
bases
- Gel electrophoresis can be used to detect the presence of different alleles if there is a difference
in the length of the DNA after digestion with RE
- Alternatively, if 2 different proteins are formed, gel electrophoresis can also be used to detect
their presence
For example, point mutation in sickle-cell anaemia causes Mst II, a RE to no longer recognize the
restriction site. No restriction takes place, generating 1 instead of 2 fragments.

(i) Outline the process of nucleic acid hybridisation and explain how it can be used to detect and analyse
restriction fragment length polymorphism (RFLP).

Nucleic Acid Hybridisation: Re-annealing of 2 single stranded nucleic acid chains of complementary base
sequence by forming hydrogen bonds, resulting in a double-stranded hybrid

Technique: Southern Blotting

- DNA cut by RE and separated by electrophoresis.
- Gel slab placed in a mixture of alkali and salt to denture the DNA fragments. dsDNA
ssDNA
- Gel placed under nitrocellulose membrane and a stack of absorbent paper.
- Capillary action draws ssDNA from gel onto nitrocellulose filter, binding in the exact position as in
the gel.
o
- Nitrocellulose filter baked at 80 C for DNA to become permanently bound to it then incubated
with radioactive DNA probe, (ssDNA synthesized to be complementary to specific nucleotide
sequences)
- Fragments containing these specific nucleotide sequences will hybridize to probe by
complementary base-pairing.
- Membrane is washed to remove unbound probes
- Autoradiography performed by placing an X-ray film over the membrane. Radioactivity of bound
probes exposes film to form dark image corresponding to bands where desired DNA-probe hybrid
can be found

Restricition Fragment Length Polymorphism

- Technique used to identify regions of DNA that may contain a gene mutation, since no 2
individuals have exact same DNA sequence (differences known as DNA polymorphisms)
- DNA polymorphism can be inherited in a co-dominant manner, present in either coding (forming
different proteins) or non-coding region
- Studied by use of restriction enzymes, assuming there is a difference in restriction fragment
length after digestion by these enzymes due to inherited differences in sites for restriction
enzymes (caused by base changes in target site such that restriction sites are lost or created)
- Hence, RFLP also refers to genetic variation in restriction fragment lengths within a population
that are produced when chromosomes are cut with a particular RE.

Using Nucleic Acid Hybridisation to Detect and Analyse RFLP

- Entire human genome is large, results in too many fragments of DNA in different sizes that
appears as a smear on the gel
- Synthetic DNA probe is used to identify the target DNA strand by complementary base pairing
(hybridisation process, through Southern Blotting)

(j) Explain how RFLP analysis facilitated the process of genomic mapping, diseases detection, DNA
fingerprinting, etc.

1. In disease detection
- Normal allele and disease causing allele can be differentiated by a difference in length of their
restriction fragments (eg. Spontaneous point mutation in sickle-cell anaemia)
 - By carrying out Southern Blotting, we can detect the presence of disease allele through
characteristic banding pattern that results on the electrophoresis gel.
- Alternatively, if sequence of alleles not known, the disease causing allele can still be identified if it
is tightly linked to a RFLP marker (both inherited as one unit)
- Limitations:
Not useful when:
More than one mutation may have caused that disease
Gene has yet been discovered for a disease
Disease caused by multiple gene interactions

2. In DNA fingerprinting
- Makes use of DNA polymorphism in non-coding regions, analyse their different lengths obtained
after digestion with restriction enzymes
- Study short repeating sequences, minisatellites (Variable Number of Tandem Repeats, VNTR) or
microsatellites (Short Tandem Repeats, STR). These vary in numbers in individuals.
- The number of repeats is heritable, thus by comparing banding patterns after restriction enzyme
cleavage, we can determine how closely related between individuals (Note: cannot exactly
pinpoint due to genetic variation and if too few bands are compared)

3. In Genomic Mapping
- Genomic mapping involves building a “picture” of the arrangement of genes and other genetic
markers relative to each other in the genome.
- Construct a genetic linkage map showing arrangement of genes and genetic markers along the
chromosomes. Their positions are calculated by cross-over values of RFLPs.
- RFLP markers are inherited in a Mendelian fashion, the higher the frequency of 2 RFLP being
inherited together, the closer the 2 loci are.

(k) Discus the goals and implications of the human genome project, including the benefits and difficult
ethical concerns.

(see 08/P3/Q4 ans)