DNA MOLECULAR TECHNIQUES
H92A 35
Outcome 4: Describe the molecular technology of DNA
sequencing, microarray analysis, and whole genome
sequencing
DNA Molecular Techniques: Outcome 4
C. Kyne; GCC; 2020
2
DNA Molecular Techniques: Outcome 4
Sequencing DNA
DNA sequencing refers to methods that determine the order (i.e. sequence) of
nucleotides in a molecule of DNA. Many methods for DNA sequencing exist but
‘Sanger sequencing’ was one of the first techniques developed for sequencing
short DNA fragments (> 600 bp). It is also known as the ‘chain termination’ method
and variants of this technology are still used today for:
•
Single gene studies e.g. identifying disease-causing mutant genes
•
Genotyping microsatellite markers
•
Validating results from other/new sequencing methods
•
Verifying plasmid sequences, inserts, mutations
•
Human leukocyte antibody (HLA) typing
During Sanger sequencing, the DNA to be sequenced acts as the template for DNA
synthesis. In fact, the requirements and procedure for Sanger sequencing are
similar to those for PCR (outcome 3). For instance, Sanger sequencing requires that
a short region (within ~1,000 base pairs) that flanks the 3’ end of the sequence of
interest is known before the experiment. From this, a synthetic DNA primer of
complementary sequence to one of the strands can be made. The primer can
anneal by complimentary base pairing to its recognition site and DNA polymerase
will extend the 3’ end of the primer to form a new single DNA strand.
From the above description, it is apparent that the following are necessary for
Sanger sequencing:
•
template DNA
•
DNA polymerase
C. Kyne; GCC; 2020
3
DNA Molecular Techniques: Outcome 4
•
primer
•
dNTPs
Another key component of the mixture is:
•
ddNTPs
which stands for di-deoxynucleotidetriphosphates and includes the bases ddATP,
ddTTP, ddGTP and ddCTP (Figure 1). These are analogues of dNTPs that terminate
chain elongation. They differ from dNTPs in that the 3'-hydroxyl is substituted with
a hydrogen (Figure 1A). Since the 3'-hydroxyl is essential for phosphodiester bond
formation and chain elongation, DNA polymerase cannot add further nucleotides
to a ddNTP because it lacks 3'-hydroxyl group. In this way, DNA synthesis is
terminated as soon as a ddNTP becomes incorporated into the chain. Sanger
sequencing takes advantage of this process and tends to use ddNTPs modified to
contain a fluorescent dye (Figure 1B). Each ddNTP has a unique excitation and
emission wavelengths, making the corresponding
nucleotide easy to detect.
During the synthesis step of Sanger sequencing, one of the target DNA strands is
incubated with: a labelled primer (complementary to the known 3’ end of the
template), DNA polymerase, dNTPs, and ddNTPs. The mixture is then subjected to
the denaturation, annealing, and extension steps as per a standard PCR experiment.
Note that the concentration of dNTPs is much higher than the concentration of
ddNTPs. This means that there is a much higher probability of a dNTP molecule
being incorporated into a newly synthesized chain than a ddNTP.
C. Kyne; GCC; 2020
4
DNA Molecular Techniques: Outcome 4
DNA synthesis of a new strand will therefore proceed until a ddNTP becomes
incorporated. Given enough time, reagents, and a suitable ratio of dNTPs to
ddNTPs, at least one DNA strand of every possible length will be produced with a
fluorescent ddNTP at the end.
Figure 1A. Comparison of the general structures of ddNTPs (top) and dNTPs (bottom) where the
former lacks a 3’ hydroxyl group. 1B. Examples of fluorescently labelled ddGTP, ddATP, ddCTP, and
ddTTs that could be used for Sanger sequencing.
After the synthesis step, each strand is separated on the basis of size using capillary
gel electrophoresis. This method uses an electric field to drive DNA through a
capillary fibre filled with a gel matrix. This method is sensitive enough to separate
DNA fragments that differ in length by a single nucleotide. The smallest fragment
will reach the end of the capillary fibre first. A fluorescencedetecting laser, built into
the machine, then shoots through the fibre and will excite the fluorescentlylabelled terminal ddNTP of the smallest fragment. The terminal ddNTP can be
identified by measuring the wavelength of the emitted fluorescence. The next
shortest fragment will then reach the fluorescencedetecting laser and will undergo
C. Kyne; GCC; 2020
5
DNA Molecular Techniques: Outcome 4
the same analysis, followed by the next shortest fragment etc. Through this process
the sequence of a full length DNA strand is determined.
The sequence of the detected fluorescence is then converted computationally into
an electropherogram (Figure 2). This is a graph of the intensity of fluorescence
emission over time which shows the nucleotide sequence above.
Figure 2. A high quality electropherogram. The data for the first 30 bases is unrealiable because of
the small fragment size.
An animation of automated Sanger sequencing can be found here
Mutation Detection by Sanger Sequencing
Sanger sequencing is a widely used method for the detection of mutations,
especially for single nucleotide variants (SNVs) which occur when a single
nucleotide is altered in a DNA sequence (Figure 3). The majority of known diseasecausing mutations occur as a result of SNVs. Studies of these SNVs by sequencing
can therefore indicate differences in susceptibility to many diseases (e.g. sickle-cell
anaemia, cystic fibrosis, and β-thalassemia). Knowledge of these genetic variations
can indicate the severity of the illness that an individual experiences as well as the
way the body responds to treatments.
Sanger sequencing analysis for mutation detection is based on a comparison: a
patient’s electropherogram is compared to that obtained from a DNA sample
C. Kyne; GCC; 2020
6
DNA Molecular Techniques: Outcome 4
without a mutation (Figure 3). Any differences between the two sequences are
analysed for their potential phenotypic effect.
Historically a visual comparison was made for each nucleotide peak in the two
traces, but this is time consuming, prone to error and slow. Nowadays, software is
used to perform sequencing analysis. Tens of thousands of nucleotides can be
analysed in seconds. The software automatically and accurately detects mutations
and provides a description of the mutation at the DNA and protein level.
Figure 3A. Electropherogram portion from a healthy individual compared to B a congenital
glaucoma patient. The black arrows highlight SNVs. Note that the mutations are heterozygous.
Mutation Detection by Microarray
Microarray technologies can also be used for the detection of gene mutations. A
microarray is also known as a “DNA chip” or a “biochip”. It is a small glass plate/slide
C. Kyne; GCC; 2020
7
DNA Molecular Techniques: Outcome 4
encased in plastic (Figure 4). Each chip contains thousands of microscopic spots
arranged in ordered rows and columns. The precise location and composition of
each spot is recorded in database. Each spot contains multiple strands of identical,
single-stranded DNA (called “probe” DNA) that represents a portion of a gene. This
means that the composition of each spot is unique. Together each portion would
add up to the normal gene in question. Other spots may contain regions of the
gene that contains previously identified mutations.
Figure 4. A DNA microarray
To identify whether a patient carries a mutation for a specific disease, a sample of
DNA from the patient's blood is collected as well as a control sample that does not
contain a mutation in the gene of interest. Both samples are prepared separately
but undergo identical processes.
The DNA is denatured in order to separate the two complementary strands of DNA.
The single-stranded molecules are cut into smaller fragments and subsequently
labelled with a fluorescent dye. Different labels are used for the patient and control
DNA. For instance, patient’s DNA could be labeled with green dye and the control
DNA could be labeled with red dye. Both sets of labeled DNA are then mixed and
inserted into the chip. They are incubated to allow for hybridization to occur
C. Kyne; GCC; 2020
8
DNA Molecular Techniques: Outcome 4
between complementary strands in the sample and on the chip. After this, excess
sample DNA is washed off the chip. DNA hybridization on the chip can be identified
by fluorescence measurements (Figure 5), which are performed using a microarray
scanner. This instrument is equipped with an autoloader and can analyze large
numbers of chips automatically.
Figure 5. Comparison of microarray data where a ☼ symbol indicates fluorescence detection due
to binding/hybridization between control/patient and probe DNA. Note that the patient and control
samples were not premixed. Instead, both samples were individually introduced to separate, but
identical chips.
If the patient lacks a mutation for the gene then both the red (control) and green
(patient) samples will bind to the sequences on the chip that represent the normal
sequence (without the mutation). Because the sample DNA is fluorescently labelled
both the red and the green dye can be independently measured, even though they
C. Kyne; GCC; 2020
9
DNA Molecular Techniques: Outcome 4
are found in the same spots. No fluorescence will be detected in the spots that
contain mutated fragments.
If the patient possesses a mutation, their DNA will not bind to the DNA sequences
on the chip that represent the "normal" DNA and no green fluorescence will
therefore be detected in those spots. Instead the mutant DNA will bind to the
sequence on the chip that represents the mutated DNA sequence
(if known), and green fluorescence will be observed in the corresponding spot.
Figure 5, shows microarray data relating to a specific gene from normal (control)
and patient DNA. Note that the patient and control samples were not premixed in
this experiment. Instead, both samples were individually incubated on separate, but
identical chips. Fluorescence was observed in position D3 and A4 of the control
chip but not the patient chip. The presence of binding between the probe DNA
(immobilized on the chip) and control DNA suggests that the ‘normal’ gene
contains a portion that is complementary to the probe DNA in D3 and A4. The lack
of binding in positions D3 and A4 of the patient sample indicates that there is a
mutation in the corresponding gene portion of the patient.
Fluorescence was not observed in position E7 of the control microarray but was
observed in the corresponding patient microarray. If E7 contains mutant DNA then
this would confirm the presence of that mutation in the patient.
Microarrays for Studies of Gene Expression
Microarrays can also be used to study the expression levels of genes in cell and
tissue samples, making it especially useful in the field of clinical diagnostics. This
process is called “gene expression profiling”. Because DNA is transcribed into
mRNA, mRNA can be used as an indicator of gene expression. The isolation of
C. Kyne; GCC; 2020
10
DNA Molecular Techniques: Outcome 4
mRNA from tissue is therefore the first step in gene expression profiling. One
suitable method of mRNA isolation from cell lysates would be to use affinity
chromatography with oligo dT beads. mRNA cannot be used directly for microarray
studies because microarrays contain single stranded DNA probes. Thus, reverse
transcription is necessary to produce cDNA from the mRNA (Outcome 2). After
amplification by PCR and fluorescent labelling, the cDNA can be hybridized on the
microarray and analysed by fluorescence. Note that the target DNA immobilized in
microarray spots are single stranded cDNA molecules that correspond to a large
number of different mRNAs. Thus each spot represents a particular gene.
C. Kyne; GCC; 2020
11
DNA Molecular Techniques: Outcome 4
Figure 6. Experimental workflow for microarray analysis of control “healthy” cells versus cancer cells.
In this example, both control and cancer cDNA was labelled with identical fluorophores and
analysed on separate, identical microarray chips. Alternatively, control and cancer cDNA could be
labelled with different fluorophores before mixing and analysing using a single microarray chip.
C. Kyne; GCC; 2020
12
DNA Molecular Techniques: Outcome 4
Consider the microarray data below (Figure 7), obtained from healthy brain cells
and brain tumour cells. From the data, it is apparent that the genes in position
D2, G4, and F6 are expressed only in healthy cells while the genes in B1, A2, and I7
are expressed only in tumour cells.
Figure 7. Comparison of microarray data where a ☼ symbol indicates fluorescence detection due
to binding/hybridization between control or tumour and probe DNA.
The method of analysis is closely similar to that used for the data in figure 5 but
the hybridization of short DNA fragments gave rise to the fluorescence data in
figure 5 while the hybridization of much longer cDNA molecules, corresponding to
expressed genes, produced the hybridization data in figure 7. The former is used
C. Kyne; GCC; 2020
13
DNA Molecular Techniques: Outcome 4
to detect mutations (Figure 5), the latter is used to detect gene expression (Figure
7).
An informative video on gene expression analysis using microarray technology can
be found here.
Gene Linkage and Recombination
Human chromosomes contain hundreds to thousands of genes each. When genes
are located close together on the same chromosome we call them “linked” (Figure
8). Linkage therefore refers to the situation in which alleles located on the same
chromosome will be inherited together as a unit more frequently than not. Alleles
located on the same chromosome are not always inherited together as a unit,
however. Recombination occurs, during the first phase of meiosis, when portions
of DNA in a homologous chromosome pair crossover, break, and recombine to
produce new combinations of alleles (Figure 8). Recombination can therefore
disrupt linked genes. Crossovers occur at random positions along the
chromosome. The frequency of crossovers between two genes is therefore
dependent on the distance between them. Short distances between allelles,
constitute a very small target for crossover events. Very few of these events will
take place. The further apart alleles are on the same chromosome, the greater the
likelihood of them undergoing recombination. These alleles would have a greater
recombination frequency than alleles located close together on the same
chromosome.
C. Kyne; GCC; 2020
14
DNA Molecular Techniques: Outcome 4
Figure 8. A, B, and C genes are partially linked. Examples of two possible recombination events are
presented. During prophase I (meiosis) crossing over occurs (at points called ‘chiasmata’) between
the maternal and paternal versions of the same chromosome, resulting in the physical exchange of
chromosome parts. This is recombination.
Recombination
contributes
to
genetic
diversity.
Additionally,
because
recombination frequency is related to the physical distance between alleles,
quantitative studies of recombination frequencies are also used to estimate the
distance between genes and to produce a type of genetic map. This process is
called “genetic mapping” or “linkage mapping” and is crucially important in
genetics and, as we will find later, genome sequencing.
Consider a simple genetic mapping study of the fruit fly Drosophila melanogaster,
which is a useful model for genetic studies. This process would begin with linkage
analysis, an experiment that quantifies the recombination frequency between a
series of gene pairs (or other genetic markers) to identify whether they are linked.
This experiment would involve analyzing the frequency at which the corresponding
phenotypes occur in a population of offspring of specific genetic crosses. High
recombination frequencies indicate a greater the distance between the genes.
C. Kyne; GCC; 2020
15
DNA Molecular Techniques: Outcome 4
Let’s consider two Drosophila genes:
• the black gene, with a dominant b+ allele that specifies normal, yellow brown
body colour and recessive b allele that specifies a black body
• The vestigial gene, which has a dominant vg+ allele that specifies normal,
long wings and a recessive vg allele that specifies short, "vestigial" wings
that are crumpled
In order to measure recombination frequency between these two genes, a fly must
be constructed in which gene recombination can be observed. The researcher must
know specifically which genes are together on the chromosome. A good way to
begin is by crossing two homozygous flies i.e. each fly has two of the same allele
for body colour and wing type (Figure 9). The resulting double heterozygote
offspring has a normal appearance and is a useful starting point for recombination
studies because it provides knowledge of which alleles are located on the same
chromosome.
Figure 9. The generation of a test fly that is heterozygous for the black and vestigial genes (F 1) by
crossing homozygous parents (P).
C. Kyne; GCC; 2020
16
DNA Molecular Techniques: Outcome 4
The double heterozygote (Figure 9; F1) is then crossed with a fly that is homozygous
recessive for body colour and wing type (it contains only b and vg alleles). This is
known as a “test cross” because it guarantees that the alleles provided by the
double heterozygote fully determine the phenotype of the
offspring. This is because the tester fly can only provide recessive alleles.
Figure 10 shows all possible offspring phenotypes produced by the test cross
including new allele combinations resulting from all possible recombination events.
The recombination frequency (RF) between these two genes can be calculated by
adding the number of individuals in the population that have non parental
phenotypes (those with black bodies and normal wings plus those with yellowbrown bodies and vestigial wings), dividing the answer by the total number of
individuals in the population, and multiplying by 100. In this case, the
recombination frequency is 17%. This means that these two genes recombine
17% of the time.
C. Kyne; GCC; 2020
17
DNA Molecular Techniques: Outcome 4
Figure 10. Results of the test cross between the double heterozygous fly and a homozygous
recessive fly. The non-parental phenotypes, which occur due to recombination, are highlighted.
Other cross tests can be performed to identify recombination frequencies for other
gene pairs. For instance, the cinnabar gene, cn, for eye pigmentation has a
recombination frequency of 8% with respect to vestigial wings. If this information
were to be presented in a map, there are two possibilities for the position of the cn
allele (Figure 11). This is because frequencies are roughly additive i.e. correlate with
the fact that genes are arranged in a linear order on a chromosome.
C. Kyne; GCC; 2020
18
DNA Molecular Techniques: Outcome 4
Figure 11. Using knowledge of the recombination frequencies of the black and cinnabar genes with
respect to the vestigial gene, two potential maps, A and B, can be generated.
The only way to confirm which of the above maps is correct is to measure the
recombination frequency for the black and cinnabar genes. Map A is correct if the
recombination frequency is ~ 25%, while map B is correct if the recombination
frequency is ~ 9%. Through experimentation, the recombination frequency for the
black and cinnabar genes was determined to be ~ 9%, making map B correct
(Figure 9).
Similarly the lobe mutation, l (which affects eye structure) was found to have a 5%
recombination frequency from the vestigial gene and a 22% recombination
frequency from the black gene. Because recombination frequencies are additive,
and we can see that 5% + 17% = 22%, this suggests that the l gene does not lie
between the black and vestigial genes, it is found on the “far” side of the vestigial
gene. Thus, the map can be updated (Figure 12).
C. Kyne; GCC; 2020
19
DNA Molecular Techniques: Outcome 4
Figure 12. Map of a Drosophila chromosome which identifies the relative position of the black,
cinnabar, vestigial, and lobe genes.
It is worth noting that linkage maps display the relative position of genes using
standard map units or percentages (1% = 1 map unit or m.u.). It is not a direct
measure of the physical distance between genes, it merely provides an
approximation of physical distance. Linkage maps do not specify the exact loci on
a chromosome or which specific chromosome they are on. Sequencing is necessary
in order to produce a physical map.
This video presents a worked example of linkage mapping.
Uses of Linkage Mapping
Linkage mapping is primarily used to understand the genetic basis of disease. For
instance, linkage mapping could be used to identify a well-known gene or marker
that is linked to a disease-causing allele. If a marker is identified, then knowledge
of the chromosome on which the disease-causing allele is located will be obtained.
Its precise location on the chromosome may also be identified. This information
will form the basis for further studies e.g. sequencing.
Linkage maps are also crucially important in genome sequencing. Draft genomes
produced through sequencing are actually composed of thousands of individual
sequences that must be pieced together to yield the final genome. Often,
sequencing alone does not provide information on how these pieces are
C. Kyne; GCC; 2020
20
DNA Molecular Techniques: Outcome 4
assembled into chromosomes. At this point, linkage maps act as frameworks for
genome assembly. Linkage mapping is also used to validate/correct mistakes in
genome sequencing efforts.
Microsatellite Markers
A genetic marker, or marker, is a gene or other DNA sequence with a known
location on a chromosome. It can therefore act as a “landmark” and be used for
identification purposes. Genes are not the only class of marker, however.
Microsatellites are tracts of DNA that are 1-10 nucleotides in length. These
nucleotide tracts repeat from 5-50 times in a row making them an example of
“tandem repeats” (Figure 13).
Figure 13. Portion of an electropherogram highlighting a microsatellite.
Microsatellites occur at thousands of locations within a genome (frequently in noncoding DNA making them biologically silent) and the physical locations of many
microsatellites are precisely known. This property makes microsatellites important
markers and analytical tools.
Specific applications of microsatellite markers include:
•
•
paternity tests
forensics
C. Kyne; GCC; 2020
21
DNA Molecular Techniques: Outcome 4
•
linkage mapping
Paternity tests: A child is likely to have similar microsatellites to their mother and
father but distinctly different microsatellites to non-relatives. This is because
microsatellites have a much higher mutation rate compared to coding DNA
sequences and therefore tend to show high variation between individuals. In
particular, the number of unit repeats tends to vary meaning that some individuals
have longer tandem repeats than others. This makes microsatellites excellent
markers for paternity identification. In a typical paternity test, hair or saliva samples
are taken from the mother, child, and proposed fathers. From the DNA in each
sample, multiple microsatellites are amplified by PCR for separation and detection
by agarose gel or capillary gel electrophoresis. The data are compared to identify
paternity (Figure 14).
Figure 14. Agarose gel electrophoresis of selected, amplified microsatellites prepared from DNA
samples of a mother, child and two potential fathers: Charles and Morgan. Morgan is the father.
Forensics: DNA profiles such as those for in paternity testing can be generated by
amplifying and separating a set of microsatellites from DNA gathered at a crime
scene. DNA from suspects can be processed in the same way and
C. Kyne; GCC; 2020
22
DNA Molecular Techniques: Outcome 4
compared in order to help identify the perpetrator.
Linkage mapping: See notes above on “Uses of Linkage Mapping”.
The Human Genome Project
The Human Genome Project (HGP) was an international project that produced a
fully sequenced human genome that is freely available in online databases. This
sequence is a prototypical or “composite” genome of several individuals. Today,
the HGP is still the world's largest collaborative biological project.
The main aims of the HGP were to:
•
determine an accurate sequence of the 3 billion DNA base pairs that
comprise the human genome
•
develop new tools for data acquisition and analysis
•
catalogue all of the estimated 20,000 to 25,000 genes in the human genome
•
sequence the genomes of other model organisms of medical relevance,
e.g. fruit fly and mouse
•
investigate the consequences of genomic research through its Ethical,
Legal, and Social Implications (ELSI) programme.
The project officially began in 1990 and by 2003 essentially all of the aims had been
met.
For more information on the Human Genome Project and its biological and
technological implications, read this article.
C. Kyne; GCC; 2020
23
DNA Molecular Techniques: Outcome 4
Sequencing in the HGP
One major goal of the HGP was to develop new technologies and analytical tools.
In particular, Sanger sequencing, which was limited to small DNA fragments, was
considered too cumbersome, expensive, and inefficient for use in such a large scale
and complex project. However, a modified version of Sanger sequencing was
deployed in the HGP. It is called BAC-to-BAC sequencing (Figure 15). The first step
of BAC-to-BAC sequencing involves generating a physical map of each
chromosome. A physical map requires breaking chromosomes into large fragments
and deciphering the order of these fragments in the intact chromosome. No
sequencing is required in this step. Instead many copies of the chromosome are
randomly cut into fragments of ~150,000 bp in length (Figure
15A). The fragments are then inserted into bacterial artificial chromosomes (BACs).
This allows the fragments to be amplified and fingerprinted i.e. analysed for the
presence of markers (in this case restriction recognition sites) that could later be
used for identification and genome assembly. For instance, some fragments
contain common portions (recall that many copies of the same chromosome are
randomly fragmented before insertion into BACs) and
identification of the restriction sites either side of a common marker can help to
piece fragments together in the correct order.
Each BAC fragment is then further fragmented into 1,500 bp pieces and placed in
another vector called M13 (Figure 15B). This clone is then sequenced. Since the
sequence of the M13 vector is already known the fragment sequence is easily
identified. The fragment sequences are assembled in the correct order using a
computer program which identifies common sequences i.e. regions of overlap
(Figure 15C). Once each 150,000 bp fragment is sequenced fully then these
fragments can be correctly ordered using the physical map.
C. Kyne; GCC; 2020
24
DNA Molecular Techniques: Outcome 4
Figure 15. Schematic of BAC-to-BAC sequencing, an example of a clone-by-clone sequencing
technique.
Whole Genome Shotgun Sequencing
During the course of the HGP advances were made in another method of
sequencing: whole genome shotgun sequencing. This method is much quicker than
the BAC-to-BAC approach because it bypasses the need to insert fragments into
BAC vectors in order to build a physical map. Whole genome shotgun sequencing
is also less expensive and requires much less starting material.
During whole genome shotgun sequencing multiple copies of the genome are
randomly fragmented into ~ 2,000 base pairs (bp) fragments. This is performed by
squeezing DNA through a pressurized syringe. Using a similar approach, a fresh
C. Kyne; GCC; 2020
25
DNA Molecular Techniques: Outcome 4
sample is fragmented into 10,000 bp pieces. Each fragment (both 2,000 and 10,000
bp types) is inserted into a plasmid. Both resulting 2,000 and 10,000 bp libraries
are
sequenced.
The
millions
of
sequenced
fragments
are
assembled
computationally into longer fragments called “contigs” and finally into a
continuous stretch of DNA corresponding to each chromosome.
To understand how the Human Genome Project has changed genetic research,
read the following article.
C. Kyne; GCC; 2020
26
DNA Molecular Techniques: Outcome 4
Outcome 4: Revision Questions
1. Give two uses for Sanger sequencing.
Single Gene Studies and verifying plasmid sequences
(2 marks)
2. List the components used in a Sanger sequencing reaction. Which component is
not required in a typical PCR reaction? Describe the role of this component.
Template DNA, DNA polymerase, primer and dNTPs, ddNTPs. ddNTPs are not
used in typical PCR reactions because 3’ – hydroxyl that is involved in dNTPs are
essential in PCR as 3’ – hydroxyl is essential for the phosphodiester bond that is
formed during the chain elongation step that is done by DNA polymerase. In
ddNTPs, the 3’ – hydroxyl group is substituted by hydrogen. The ddNTPs are
used in Sanger sequencing as they can be easily modified to contain a
fluorescent dye.
(7 marks)
3. Describe the Sanger method of DNA sequencing.





One of the DNA strands is incubated with a labelled primer
(complimentary to the 3’ end of the template), DNA polymerase, dNTPs,
and ddNTPS.
The mixture is then exposed to the steps of PCR; denaturing, annealing
and extension. (The concentration of dNTPs is higher than the
concentration of ddNTPs)
Due to there being a higher concentration of dNTPs, there is a higher
chance of dNTPs getting incorporated into the newly synthesised chain.
DNA synthesis will continue with the new strand until a ddNTP is
incorporated into the strand.
Over time, if there are enough reagents and suitable ratios of dNTPs to
ddNTPs, then at least one DNA strand of every possible length will
produce a fluorescent ddNTP at their end.
C. Kyne; GCC; 2020
27
DNA Molecular Techniques: Outcome 4
(6 marks)
4. Explain how the newly synthesized DNA fragments are separated and detected
during Sanger sequencing.
The new strands are separated on the criteria of size using capillary gel
electrophoresis. Electric fields are used to drive DNA through the capillary fibre
filled with a gel matrix. The method is sensitive enough to separate DNA
fragments that differ in length by a single nucleotide. The smallest fragment will
reach the end of the complex first and then a fluorescent laser built into the
machine shoot through the complex to excite the fluorescent markers of the
smallest fragment. The ddNTP can be identified doing this to measure the
wavelength of the emitted fluorescence. Through repeating this process with the
varying DNA sample sizes, the full sequence of the DNA strand can be
determined.
(2 marks)
5. What is an electropherogram?
A machine that detects fluorescent waves admitted by ddNTPs to plot a
wavelength to show the length of DNA fragments to fully determine the length
of the synthesised DNA strand.
(1 mark)
6. Analyse the electropherogram below. Describe the type of mutation that is
apparent.
Most of the DNA bases presents on both of the waves are similar, however there
are very slight variations between the wave sizes can be seen between the C
bases. The most prominent point mutation is between the blue C bases. The
C. Kyne; GCC; 2020
28
DNA Molecular Techniques: Outcome 4
wave is much shorter and is the most prominent difference between the 2 waves.
The most apparent mutation is point mutation.
(2 marks)
7. DNA microarrays can be used for the identification of mutations and for studies
of gene expression. Explain the features of a DNA microarray.
DNA microarrays are also known as a DNA chips. They are a small plate/ slide
that are encased in plastic. The chips contain thousands of microscopic spots
that are arranged in ordered rows and columns. Their precise locations are
entered into a data base. Each of the spots contain multiple strands of identical,
single stranded DNA that represent a portion of the gene. Each composition of
the spot is unique. Each of the spots would add up to a normal gene.
(4 marks)
8. Explain how a tissue sample must be prepared/processed for a microarray-based
study of gene expression.
C. Kyne; GCC; 2020
29
DNA Molecular Techniques: Outcome 4
The patient’s blood sample and a control sample with no mutations are taken
separately. The DNA in both samples is denatured in order to separate the 2
complimentary strands of DNA. The single stranded molecules are then cut into
smaller fragments and then labelled with a fluorescent dye. Various labels are
used for the patient and control DNA. Both sets of labelled DNA are mixed and
inserted into a chip. Then they are incubated to allow for hybridisation to occur
between the complimentary strands in the sample and on the chip. After this,
the excess sample DNA is then washed off the chi. The DNA hybridisation on the
chip can be identified by the fluorescent measurements which are done by using
a microarray scanner.
(4 marks)
9. The scheme below shows some of the genes expressed in healthy pancreatic
tissue and a pancreatic cancer cells.
C. Kyne; GCC; 2020
30
DNA Molecular Techniques: Outcome 4
(a) Which is the control experiment?
Healthy Pancreatic tissue sample
(1 mark)
(b) Identify a well/spot that contains a gene that is only expressed in healthy
pancreatic cells.
A2
(1 mark)
(c) Identify a well/spot that contains a gene that is only expressed in pancreatic
cancer cells.
B3
C. Kyne; GCC; 2020
31
DNA Molecular Techniques: Outcome 4
(1 mark)
10. What process is responsible for disrupting linked genes during meiosis?
Recombination
(1 mark)
11. What is meant by the term “recombination frequency”?
The closer together the alleles are on the chromosomes, the greater the chance
of recombination than those that are further away.
(1 mark)
12. What is a linkage map and how does linkage mapping work?
A linkage map shows the distance between 2 chromosomes to help estimate
recombination frequency. Recombination frequency is linked to the distance
between alleles. By studying the distance between recombination frequencies,
it can be used to plot a linkage map.
(2 marks)
13. Describe two applications of linkage mapping in genomic science.
Could be used to identify a well-known gene of marker that could be linked to
a disease-causing allele. They are also important in genome sequencing.
(2 marks)
14. What is the difference between a linkage map and a physical map?
Linkage maps represent the distance between alleles to represent
recombination frequencies whilst physical maps are used to identify the order
of large fragments before any sequencing can occur.
(1 mark)
C. Kyne; GCC; 2020
32
DNA Molecular Techniques: Outcome 4
15. Genes A, B, C, and D are located on the same chromosome. The recombination
frequencies (RF) are as follows:
Genes RF (%)
A-B
10
A-C
25
A-D
23
B-C
15
C-D
48
What is the most likely order of genes in the chromosome?
C,B,A,D
(2 marks)
16. Genes A, B, G, and H are located on the same chromosome. The distances
between the genes are below:
Genes RF (%)
A-H
18
A-B
10
B-H
8
A-G
2
H-G
20
What is the most likely order of genes in the chromosome?
H,B,A,G
(2 marks)
C. Kyne; GCC; 2020
33
DNA Molecular Techniques: Outcome 4
17. What is a microsatellite marker?
These are tracts of DNA that are 1-10 nucleotides in length. They occur in
thousands of locations within the genome and the physical locations of many
of these satellites are used as genomic markers for identical purposes.
(2 marks)
18. Describe how microsatellites are used in either:
•
paternity testing; or
•
forensics
Paternity Test: A child will have similar microsatellites to their parents than
to their non- relatives. Microsatellites have a higher rate of mutation than
coding DNA sequences therefore it shows a higher variation between
individuals. This makes microsatellites perfect for paternity tests as the
number of repeating units tends to vary in some individuals resulting in
longer tandem repeats than others. A sample from the child is then
compared to the mother and the proposed father. From each of the DNA in
the sample, multiple microsatellites are then amplified by PCR for separation
and then under detection by either an agarose gel or capillary gel
electrophoresis.
Forensics: DNA profiles can be generated by amplifying and separating a
set of microsatellites from DNA gathered at the crime scene. DNA from the
suspects can be compared to the perpetrator by the same process.
(3 marks)
19. State 4 aims of the Human Genome Project (HGP).




To determine an accurate sequence of the 3 billion DNA base pairs that
comprise the human genome
Develop new tools for data acquisition and analysis
Catalogue the genomes of other models organisms of medical relevance
Legal, and social implications program
(4 marks)
C. Kyne; GCC; 2020
34
DNA Molecular Techniques: Outcome 4
20. Describe the method of BAC-to-BAC sequencing used in the HGP.





Generate a physical map by breaking down the chromosomes into large
chromosomes into large fragments and deciphering the order of the
fragments in the intact chromosome. No sequencing is required in this step.
Instead, many copies of the chromosome are randomly cut into fragments
The fragments are inserted into bacterial artificial chromosomes, allowing
for the amplification and fingerprinted. This is used to look for the presence
of markers. These markers can later be used to for identification and
genome assembly.
Each BAC fragment is then further fragments into 1,500 bp pieces and
placed in another vector called M13. This clone is then sequenced. Since the
sequence for the M13 vector is already known, the fragment sequence is
easily identified
The fragment sequences are assembled into the correct order using a
computer program.
These fragments are ordered properly using a physical map.
(3 marks)
21. Describe how whole genome shotgun sequencing works.




Multiple copies of the genome are randomly fragmented into 2,000 base
pairs fragments. This is done by squeezing the DNA through a
pressurized syringe.
Using a similar approach, a fresh sample is fragments into 10,000 bp
pieces. These both result in 2,000 and 10,000 bp libraries being
sequenced.
The millions of fragments are assembled computationally into longer
fragments called contigs.
Then they are assembled into a continuous stretch of DNA
corresponding to each chromosome.
(3 marks)
22. Give two advantages of whole genome shotgun sequencing over BAC-to-BAC
sequencing.
C. Kyne; GCC; 2020
35
DNA Molecular Techniques: Outcome 4
Whole Genome shotgun sequencing is overall less expensive and requires
much less starting material than BAC – to – BAC sequencing.
(2 marks)
C. Kyne; GCC; 2020
36