Download Point Mutation Detection

GENETICS: Case 1 and Case 5
Adkison and Brown
Molecular Genetic Techniques
A physician should be able to interpret fundamental genetic data just as they interpret
biochemical data, ECGs, or respirometry results. To do so, an understanding of basic molecular
genetic methodologies is necessary. Below, these fundamental approaches and applications to
molecular diagnostics are described.
Nucleic Acid Visualization
Patient cells are necessary to initiate molecular genetic analysis. Typically, patient
material is a blood sample, biopsy specimen, and amniotic fluid or chorionic villus sample,
though any nucleated cell sample is acceptable. Using blood as an example, total genomic DNA
is extracted and the DNA is visualized and/or prepared for subsequent analysis by a number of
techniques including restriction fragment length polymorphism (RFLP) and Southern blotting,
DNA amplification using the polymerase chain reaction (PCR), or DNA sequence analysis.
RFLP and Southern Blot Analysis
Southern blotting is a method to visualize DNA of interest. There are three fundamental
elements to the Southern blot procedure: (a) fragmentation of the DNA, (b) separation of the
DNA on the basis of fragment size, and (c) identification and visualization of informative
fragments using a probe. Fragmentation typically occurs through the use of restriction
endonucleases, which are also referred to as restriction enzymes. Restriction endonucleases are
enzymes of bacterial origin that bind to the DNA at specific sites and cleave both strands of the
DNA. The DNA recognition sites for restriction enzymes are typically palindromic sequences 4
to 8 bases in length and the double stranded breaks occur within or adjacent to the recognition
sites. For example, the restriction enzyme EcoRI recognizes and binds to the six nucleotide
sequence of 5′-GAATTC-3′. In this case, the enzyme nicks the phosphodiester bond between the
G and the A on both strands, generating two DNA fragments with so-called “sticky ends” (Table
1). Because a six base pair sequence such as GAATTC is encountered frequently in the human
genome, treatment of genomic DNA with the EcoRI enzyme generates roughly one million DNA
fragments. A single base change in a restriction enzyme recognition site prevents an
endonuclease from binding to the DNA, resulting in no cleavage of the DNA. Conversely, a
single base change may create a new recognition site where none existed prior to the base
change. Different individuals in the population harbor a variety of benign point mutations that
alter restriction enzyme recognition sites, thus resulting in a different collection of fragments.
Such variation in the sizes of DNA fragments obtained by restriction endonuclease digestion is
visualized by gel electrophoresis and Southern blotting; this procedure is termed “restriction
fragment length polymorphism,” or RFLP, analysis (Figure 1).
For gel electrophoresis, DNA fragments are loaded onto a porous, gelatinous material to
which an electrical field is applied. By virtue of the inherent negative charge of DNA molecules,
fragments migrate to the positive pole. Smaller fragments migrate faster than larger fragments
thereby resolving in the gel on the basis of size. The fragments are visualized by the addition of a
dye molecule, such as ethidium bromide, that intercalates between the DNA base pairs and
fluoresces upon illumination with UV light. Other fluorescent dyes with greater sensitivity to
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staining small quantities of DNA and less toxicity are also used. Often however, a particular
fragment of diagnostic value is of interest and it is impossible to identify an individual fragment
with many fragments resolved in the gel. The ability to visualize single DNA fragments requires
the addition of a DNA probe in these situations that hybridizes specifically to the fragment of
interest. A DNA probe is typically single stranded and complimentary to the gene or region of
DNA interest. It is labeled with a fluorescent or radioactive tag so that the probe:target complex
can be detected. Probes can be DNA fragments purified from restriction endonucleases digestion
or small, synthetic DNA oligonucleotides are synthesized.
Table 1. Examples of Commonly Used Restriction Endonucleases. Arrows show
restriction sites of sequence.
Type of
Product
Recognition
Sequence
Blunt ends
AGCT
TCGA
5’ AG….……………..CT
3’
3’
TC………...………GA5’
EcoRI
Escherichia coli
strain R
5’ overhang
GAATTC
CTTAAG
5’ G…………….AATTC
3’
3’CTTAA….............… G
5’
PstI
Providencia
stuatii
3’overhang
CTGCAG
GACGTC
5’CTGCA.....................G
3’
3’G……….……ACGTC 5’
MnlI
Moraxella
nonliquefaciens
Nonpalindromic
sequence
Enzyme
Source
AluI
Arthrobacter
luteus
Fragment Ends
CCTCNNNNNNN 5’CCTC(N7)3’
GGAGNNNNNNN 3’GGAG(N6)...N5’
A gene-specific probe is not hybridized to its complement in an agarose gel due to the
difficulty of working with the gel matrix itself and due to any radioactive background that could
occur with this type of labeling. Instead, the fragmented DNA is transferred to a dry filter and the
probe is added. This is the essence of the Southern blot procedure (see Figure 1). Specifically,
following gel electrophoresis, the DNA strands are denatured with a strong base while still in the
gel. The single-stranded DNA is then transferred to a nylon or nitrocellulose membrane support
by capillary action; sometimes application of a vacuum facilitates transfer. At this point, the
denatured DNA is “fixed” to the membrane and a probe is applied under conditions that favor the
re-establishment of DNA duplexes. As suggested above, it is the Watson-Crick hydrogen bond
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base pairing between the single strand probe and its single-stranded complement restriction
fragment that enables the probe to specifically anneal only to its complement. Visualization of a
Figure 1. Southern Blotting technique is used to visualize fragments of variable
lengths generated by various restriction endonucleases (RFLP analysis).
Genomic DNA
Restriction
Endonuclease
Fragmented
Genomic DNA
Large
fragments
-
Small
fragments
+
DNA separated on
an agarose gel
Transfer (blot) DNA to filter
Hybridize (probe) filter with radioactive or non-radioactive
probe to gene or region of interest; remove unbound probe
Autoradiography or Development
Visualization of fragments
complementary to probe
DNA fragment of interest is performed by laser-facilitated detection of the fluorescent probe or
by exposure of the filter to X-ray film in the case of a radioactive probe. Thus, by using a
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combination of restriction endonuclease digestion, gel electrophoresis, and Southern blotting,
visualizing a DNA fragment of interest is accomplished.
Southern blotting, as described, is a useful tool for identifying DNAs. Another similar
tool is the Northern blot which has many similarities to the Southern blotting. The primary
difference is that Northern blot provide visualization of RNAs and is therefore quite useful to
determine the presence of RNAs, particularly mRNA, being expressed. The presence of mRNAs
documents that the gene is indeed expressed which also validates the presence of a functional
promoter and suggests the size of the protein. mRNAs that are smaller or larger than the
expected size provide information suggesting a deletion or insertion in the gene or abnormal
splicing. If the quantity of mRNA present is less than expected, this may suggest a weak
promoter, which can occur for several reasons, or provoke questions about the half-life of the
RNA.
The Polymerase Chain Reaction (PCR)
The introduction of polymerase chain reaction (PCR) has revolutionized DNA-based
diagnostics. The rapid, inexpensive amplification of specific DNA sequences made possible with
PCR has tremendously enabled both preparative and analytical procedures. PCR is the in vitro
enzymatic amplification of a short (up to 5-6 kb) and specific DNA sequence. Amplification can
be initiated with even a single DNA molecule and produces millions of copies in a period of a
few hours. There are four essential components to PCR: two deoxyoligonucleotide primers, a
thermostable DNA polymerase, target DNA, and nucleotides. The primers add specificity to the
amplification by defining the flanking regions of DNA sequences to be amplified. Primers are
“designed” to reflect the complimentary nucleotide sequence of the target. These are usually 15
– 30 nucleotides in length and synthesized by an automated process. Primers bind to targets in a
5’-3’ direction on each strand and amplification occurs between them.
Amplification occurs during a series of denaturing, heating, and cooling phases that are
repeated numerous times. These serve to dissociate double stranded DNA, allow primers to
anneal, and facilitate new strand extension. The polymerase used in this procedure must be
thermostable at high temperatures, a feature not characteristic of enzymes. The best known of
the special thermostable polymerases was isolated from Thermus aquaticus. Designated DNA
taq polymerase, it withstands repeated cycles of 95˚C or greater.
During “denaturation,” the target DNA in the reaction is heated to 95oC rendering the
target DNA single-stranded by breaking the hydrogen bonds between the two strands. The
reaction is then cooled, typically to 45 – 65oC, to permit “annealing” of the single-stranded
primers to complimentary sequences in the target DNA. Finally, the temperature is increased to
70-75oC to allow extension or synthesis of the new DNA most efficiently. During the extension
phase, DNA polymerase uses the free 3′-OH group on the primers to synthesize new DNA. This
3 step or phase cycle is repeated with the newly synthesized DNA strands serving as templates
for additional strand formation. Early in the process, the original genomic DNA is diluted out
and the ends of the newly synthesized DNA strands are entirely defined by the primers. Overall,
this cycle is repeated 20-35 times and the number of DNA copies doubles with each cycle, thus
resulting in millions of copies of specific, primer-directed DNA fragments (see Karp Figure
18.48). It should be noted that the thermostable DNA polymerase, an excess of primers, and the
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formation of newly synthesized DNA fragments that serve as templates for subsequent cycles
permit the iterative cycles of amplification.
Application of the PCR in molecular genetic diagnostics will be described below for
detecting mutations. It should be apparent, however, that PCR has two direct utilities. First, it can
provide abundant substrate for mutation detection strategies. Second, it can be a stand-alone
diagnostic tool for detecting changes in DNA length, such as small insertions/deletions. In either
case, PCR has greatly facilitated the era of genetic medicine by providing a rapid and
inexpensive tool to biomedical scientists working on both diagnostic and research procedures
(Table 2).
Table 2. Comparison of PCR and RFLP
RFLP
Time Requirement
Amount of DNA required
Cost
Sensitivity
Days
Micrograms to milligrams
May be high with radioactivity
usage and disposal; less costly
if using non-radioactive
probes
Less sensitive to small
quantities of DNA
PCR
Hours
Picograms or less
Rarely needs radioactivity and
therefore less expensive
Most sensitive to small
quantities or copy number
DNA Sequence Analysis
In the post-Human Genome Project era, “unknown” regions of the chromosome are no
longer sequenced to find new disease-causing mutations. More typically, genes or certain regions
of genes are sequenced to detect a disease-specific mutation. Because the entire sequence of the
human genome is known, PCR primers can be designed to amplify specific DNA for sequencing.
Since DNA sequencing is greatly facilitated by abundant target DNA, only one application of
PCR amplification of a specific DNA substrate is generally needed for sequencing.
The most common method of DNA sequencing is the Sanger dideoxynucleotide chain
terminating technique (Figure 2A). Here, heat denatured, single-stranded template, such as PCR
products, is added to each of four tubes. Each tube contains a mixture of the four nucleotides (A,
G, C, and T) that acts as a substrate for DNA polymerase. Each tube also contains one of the four
chain terminating dideoxy ribonucleotides (ddA, ddG, ddC, or ddT). Hence the “A” tube
contains have a mixture of the four normal radioactively labeled nucleotides (dA, dG, dT, and
dC) plus the chain terminating ddA; a similar mixture of dNTPs to ddNTPs is found in the “G,”
“C,” and “T” tubes. The incorporation of any dideoxy nucleotides prohibits further DNA
polymerization because these lack the 3′-OH group required by DNA polymerase to add the next
nucleotide. The addition of DNA polymerase and oligonucleotide primers, which are required to
anneal to the target and initiate DNA polymerization via the free 3′-OH, to each reaction tube
initiates DNA synthesis. However, the chains are terminated at different positions due to the
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random insertion of a dideoxynucleotide instead of a normal deoxynucleotide. Stated differently,
when a ddG is inserted by the DNA polymerase instead of a dG, synthesis stops at that point. In
the “G” tube reaction, for example, some chains will be terminated at the initial G encountered
by the DNA polymerase, while other chains will be halted at other G positions of the chain. In
the “G” tube, the ratio of dGTP/ddGTP is such that there will be a number of chains that have
terminated at each G position along the template.
Figure 2. DNA sequencing. A. Sequencing done with a radioactive labeled
deoxynucleotides. [used with permission: genetics.nbii.gov/ basic2.html. Probes
may be radioactive or non-radioactive. B. Automated sequencing can be done
with fluorescent dyes attached to dideoxynucleotides.
A
B
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The four reactions are separated by gel electorphoresis on a polyacrylamide gel to
produce a visual “ladder” of DNA fragments. Labeling either the DNA sequencing primers or
the individual nucleotides with a radioactive or fluorescent tag enables this visualization. In
automated sequencing, the ddNTPs are labeled with fluorescent dyes that are detected by a
scanner. Because the gel lane containing “A” (from the “A” tube), “T” and so forth is known, it
is a straightforward matter to read the sequencing ladder.
The majority of DNA sequencing was once done by polyacrylamide gel electrophoresis
but more recently is being replaced by capillary electrophoresis (Figure 2B). This technique
adds automation to the process while adding several highly regarded features. DNA separation
occurs in a capillary with a diameter of 25-100 μm, which provides a high ratio of surface area to
volume that acts to dissipate heat produced. This feature allows the use of higher electric fields
that decreases time of separation and increases resolution of DNA.
Mutation Detection
The true basis of molecular diagnostics is the detection of specific disease-causing
mutations. Here, the most common methods associated genetic variation that are employed in
laboratories for the detection of common diseases are discussed and include point mutations,
deletions, and trinucleotide repeat expansions.
Point Mutation Detection
Mutation-specific RFLPs. In some single gene disorders, the causal mutation is a point
mutation that alters a restriction endonuclease recognition site. This type of mutation can either
abolish an existing recognition site or create a novel site. When this happens, it permits a
mutation-specific test that can be used for diagnostic purposes. Perhaps the best example of a
mutation-specific RFLP test is found in sickle cell anemia. Sickle cell anemia results from an A
to T missense mutation and the substitution of a valine for a glutamate at the sixth amino acid of
the β-globin polypeptide. This base change affects the 5′-CCTNAGG-3′ (where N can be any
nucleotide) recognition site for the MstII restriction endonuclease because the central A is
replaced by a T, rendering a similar but different 5-CCTNTGG-3′ sequence. MstII does not
recognize or cleave this altered DNA sequence. Hence, sickle cell anemia patients differ from the
normal population by the loss of this particular restriction site, resulting in a RFLP for sickle cell
anemia. In the laboratory, this is recognized with agarose gel electrophoresis where normal
individuals have two smaller DNA fragments that corresponded to the affected individual’s
single, longer DNA fragment (Figure 3). While specific and sensitive, this methodology is
relatively rare in practice because the majority of point mutations do not alter a restriction
endonucleases recognition site. As suggested earlier, PCR can also be used to amplify a
fragment of interest for restriction analysis. The fragment sizes may differ between genomic
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DNA versus PCR-generated DNA and a probe may be required to visualize a fragment in
genomic DNA, but the results still demonstrate the affect of the mutation.
Figure 3. RFLP analysis of the -globin gene and sickle cell anemia. A. An A-to-T point
mutation occurs in codon 6. B. The mutation eliminates a recognition site for MstII. Individuals
with sickle trait are heterozygous and have a 1.15 kb and 1.35 kb fragments. Individuals with
sickle cell anemia have 2 longer 1.35 kb fragments.
A
B
C
Allele specific oligonucleotides. Thousands of single base changes are associated with
human disease. This has necessitated the development of other methods to identify individual
point mutations because mutation-specific RFLPs are relatively rare. One of these methods is
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allele specific oligonucleotide (ASO) hybridization (Figure 4). For this, a DNA synthesizer
creates short, synthetic oligonucleotides that are an exact compliment of the normal DNA
sequence. These, in turn, are radioactively or fluorescently labeled and used to probe patient
and/or control DNA in a process similar to the Southern blot probing. During the hybridization
process, these short oligonucleotides only bind to a perfect sequence complement and not to any
sequence with even a single mismatched base. Thus, the oligonucleotides only bind normal
DNAs, making it simple to distinguish between normal DNA and mutant DNA. Conversely, a
short oligonucleotide may also be synthesized that is a perfect complement to a mutant DNA
sequence. For example, an ASO can be synthesized that hybridizes only to the portion of the globin gene that contains the sickle cell anemia mutation. Such an oligonucleotide does not bind
to normal DNA because it is mismatched at the base altered in sickle cell anemia. A mutationspecific ASO, then, can be used to screen patients, family members, and even members of the
general population for the presence of the sickle cell anemia mutation.
Figure 4. Allele specific oligonucleotides can be designed to identify specific
mutations either known to occur in a family or that occur at a high frequency in
the population. In this example, if the ASO are designed to the normal and
mutant region of the -globin gene, the fetus is a normal carrier rather than
affected.
Single-stranded
DNA samples from
family members
Single-stranded
DNA is made from
a normal allele (A)
GGACTCCTC
Single-stranded
DNA is made from
a mutant allele (a)
GGACTACTC
A probe is made
for each allele
CCTGAGGAG
CCTGATGAG
Probes are hybridized
to sample DNAs
Father
Child
Mother
Fetus
Probe for normal
allele (A)
Probe for mutant
allele (a)
Deduced
Genotypes
Aa
Aa AA Aa
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DNA Sequencing
In special cases where a patient is clearly suffering from a particular disease but does not
harbor known genetic variants associated with the disease, the candidate gene may be subjected
to complete or partial automated DNA sequence analysis (Figure 2). DNA sequencing is much
more labor intensive and time consuming if the specific mutation is unknown, but it is an
excellent approach for the discovery of new or rare mutations. However, this technique is so
valuable that increased technological developments are providing improvements designed to
reduce the time and cost of sequencing; it is not unthinkable that samples currently referred to a
research lab for evaluation will be sequenced in a diagnostic lab in the near future.
Deletion Detection
Very small deletions, typically single base deletions that cause disease may be screened
for using the same methodologies as employed for point mutations. Larger deletions, however,
require a different approach. PCR is ideal for detecting deletions in the 100 base pair to 4 kb
range since primer pairs amplify both a normal and mutated allele containing a deletion.
Following PCR, DNA fragments are separated by electrophoresis and compared to molecular
weight standards. Deleted alleles are obviously not as large as the non-deleted forms and hence
easily visualized.
Trinucleotide Repeat Expansion Detection
Expansions of trinucleotide sequences are a relatively common genetic mechanism for
neurological disease. Fragile X syndrome, Huntington disease, myotonic dystrophy, spinobulbar
muscular atrophy and Friedreich’s ataxia are all examples of disease caused by expanding
trinucleotide repeats. This class of mutation poses a special challenge for diagnostics as
illustrated by the Fragile X syndrome. Fragile X1 is due to an expansion of a CGG trinucleotide
in the 5′ untranslated region of the FMR1 gene. Three classes of FMR1-associated CGG
expansions are recognized in the population: normal chromosomes contain between 5 and 50
repeats; premutation chromosomes contain between 50 and 200 repeats, and full mutation
chromosomes (affected individuals) feature 200 to 2000 CGG repeats. Distinguishing between
these three allelic repeats seems, intuitively, an ideal task for PCR. In practice however, this is
true for normal and premutation alleles; PCR primers flank the site of the CGG expansion and
amplify the alleles. The same is not always true for the full mutation because full mutation
expansions can reach 6 kb in length, which exceeds the size where accurate genotyping can be
done on the basis of PCR alone. Thus, Southern blotting is typically done when Fragile X
syndrome is suspected. In this case, a probe is used that hybridizes proximal to the expanded
region. In this way, very large expansions can be detected. The dividing line between using PCR
and Southern to size the repeat tract is between 70-100 repeats. Southern blots cannot identify a
precise repeat size at the normal and low permutation range, but PCR is unable to correctly
identify repeat tracts over 70-100 units.
It is important to note that DNA diagnostics pervades the entirety of the health care
system today. A large number of laboratories utilizing genetic techniques now exist and stand as
a testament to the ubiquity of genetic defects in medicine. Even in rural or remote settings, the
physician sends blood samples to a laboratory for DNA analysis. The association between
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Fragile X syndrome will be discussed in detail for Case 8.
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medicine and molecular genetics will grow more comprehensive as additional insights are made
regarding genetic predisposition to disease and the role of genetics in pharmaceutical efficacy.
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