CLIN. CHEM.

35/9, 1843-1848 (1989)

Use of Polymerase Chain Reaction for Diagnosis of Inherited Disorders

Corlnne D. Boehm

The polymerase chain reaction (PCR) is a rapid method for CYCLE#: 1 2

% D-
3
generating a 10#{176}-
to 107-fold increase in the number of
- ,.
copies of a discrete DNA or RNA sequence. The technique is
being used for rapid prenataldiagnosisand carriertestingof Cc c3
-
several inheriteddisorders.After PCR, mutationsproducing
single-gene disorderscan be detected by several different
methods, includingendonucleasedigestionand gel electro-
phoresis (applicablewhen a mutationaffectsan endonucle-
___
-5
L
-a
*

ase recognitionsite), gel electrophoresis(used for detection 4-

of deletions), and hybridizationto an oligonucleotideprobe

specific for a mutation. Less often, gene sequencing of a
PCR product is used to rapidly identify a mutation. In ::::::i..
addition,the PCR techniquecan be appliedto polymorphism Fig. 1. Accumulationof targetsequencesduringthree cyclesof
analysis to provide diagnosis by linkage analysis. In other polymerase amplification
areas, PCR is being used to detect and characterize micro- Denatured genomic DNA strands are shown as a broken ladder in the middle
offigure. (-), products from the 1st cycle ofamplification;(- - - -), products
bial pathogens and to characterize mutations associated with from the2nd cycle; and (- - -), from the 3rd cycle. (0, U), the two primers
carcinogenesis.The PCR method is useful in situationsin delineating the boundaries ofthe target sequence. Extension products are of
variable length when genomic DNA serves as template (products with a,rowat
which the amount of DNA sample is limited, such as in one end), but of specific length when a previous extension product is the
forensicsand prenatal testing,or in which the qualityof the template
DNA sample is poor.
cent template DNA. After the first cycle, both native DNA
The polymerase chain reaction (PCR) is an enzymatic
and DNA generated from extension of the primers in
method of synthesizing millions of copies of a discrete
earlier cycles will be used as templates. The reaction is
sequence of DNA or RNA (1-4). Developed and improved
most easily and effectively carried out through use of a
over the past five years, this method has become very
machine that can be programmed to make rapid and
popular in many areas of biological and medical research
accurate temperature changes, but it can also be performed
and diagnosis. It allows simplification of many of the more
manually by moving the reaction tube between heatblocks
tedious steps involved in manipulation of genetic se-
or waterbaths of the required temperatures. Theoretically,
quences, thereby allowing research questions to be an-
the number of copies of target sequence doubles with each
swered more rapidly; in addition, it has provided novel
cycle. In practice, the number of target sequences increases
approaches to analysis of genetic sequences.
by approximately 10#{176}-
to 107-fold after 30 cycles.
Use of the technique requires knowledge of the nucleo-
The reaction as originally described used the Klenow
tide sequences that border either side of the sequence to be
fragment of Escherichia coli DNA polymerase I. However,
amplified. Oligonucleotides complementary to these two
because this enzyme is inactivated by the higher tempera-
bordering sequences are synthesized and then used in the
tures required for DNA denaturation, a fresh aliquot of this
PCR reaction. They hybridize to the bordering sequences
polyinerase must be added after the denaturation step in
and then serve as primers for the polymerase, which
each cycle. More recently, development of a thermostable
extends these primer sequences by copying the adjacent
DNA polymerase has overcome the need to re-introduce
template DNA in the 3’ direction. The pair of primers is
enzyme with each cycle. In addition to simplifying the
oriented such, that the DNA polymerase extension of each
reaction, this Taq polymerase also increases the specificity
of the pair of sequences will extend across the region
of the reaction substantially (5, 6).
between them (Figure 1).
Only very small amounts of DNA are required for the
The reaction consists of repeated cycles of temperature-
reaction. A single-copy sequence from human genomic
dependent steps. First, the double-stranded template DNA
DNA can be amplified from a sample of <50 ng of DNA.
is denatured into single strands by heating. Second, the
DNA from a single hair root, or from a single sperm, has
primer sequences bind to complementary (or nearly com-
provided sufficient target DNA for amplification and sub-
plementary) sequences; the specificity with which each one
sequent genetic analysis (7, 8). Thus the PCR reaction is
hybridizes to a sequence may be controlled by temperature
also helpful in situations in which a limited amount of
adjustments, to permit amplification of sequences flanked
DNA is available. Now that DNA fingerprinting is gaining
by regions that are similar but not identical in complemen-
recognition as a useful method for distinguishing individ-
tarity to the primers. Third, a DNA polymerase extends the
uals, obtaining DNA sequences from a limited number of
primer sequences by copying the sequence from the adja-
cells, as can be accomplished after PCR amplification,
should become an important tool in forensics (9). In addi-
tion, samples that are unsuitable for examination by con-
Johns Hopkins University School of Medicine, Department of
Pediatrics, Baltimore, MD 21205. ventional Southern blotting because of partial degradation

Received April 13, 1989; accepted June 27, 1989. of the DNA may be suitable for analysis after PCR ampli-

CLINICALCHEMISTRY,Vol.35, No.9, 1989 1843

fication (10, 11). However, one drawback of the procedure’s amino acid substitution (Glu-Val) at codon 6 of the /3-
sensitivity is that trace amounts of contaminating se- globin gene, also destroys a normal Cun I recognition site.
quence can be co-amplified, and their presence may result This change from normal in the restriction pattern of the
in genetic findings not representative of the sample being gene can be easily detected as an alternation in the DNA
examined (12). fragment size after Cun I digestion of /3-globin-amplifled
PCR technology is finding many applications, both in DNA, agarose gel electrophoresis, and staining the gel with
diagnostic and research situations. In addition to detecting ethidium bromide (52). Ethidium bromide, which interca-
mutations that cause inherited disorders, it is currently lates into DNA, fluoresces under ultraviolet light, making
also used in microbiology to identif’ viral and bacterial visible the DNA fragments within the gel. Because migra-
pathogens (13-22) and is being used to examine gene tion within the gel depends on fragment size, diagnosis can
function and regulation in cancer research (23-32).
be made by analyzing the DNA fragment pattern after
electrophoresis (Figure 2). If possible, the fragment to be
Use of PCR In DIagnosIs of InherIted DIseases
amplified should contain at least one constant recognition
The usefulness of PCR technology was first demonstrated site for the endonuclease. If the endonuclease digestion is
in the field of genetics, in which it has been used for incomplete, the full-size PCR product will still be evident
prenatal and carrier testing of several monogenic disorders on the gel after digestion and will indicate the need for a
including the hemoglobinopathies (sickle cell anemia, and repeat analysis.
/3- and a-thalassemia) (3, 33-36), Duchenne and Becker About 50% of/3-thalassemia mutations alter a restriction
muscular dystrophy (37), cystic fibrosis (38,39), hemophilia endonuclease site, either by creating a new one or destroy-
A and B (Factor Vifi and IX deficiency) (40-44), a1- ing one that is already present. These mutations can be
antitrypsin deficiency (45, 46), familial hypercholesterol- rapidly detected by digesting the amplified /3-globin se-
emia (47), apolipoprotein C-Il deficiency (48), Huntington’s quences with the appropriate endonuclease, followed by
chorea (49), and Tay-Sachs disease (50, 51). agarose gel electrophoresis (53).
Inherited variations in DNA can be detected by several
methods, and many mutations can be detected by more
than one method. All methods can be carried out with 02002
______________________________________
lv’ V/////’////.’.2Z///A vO 2 25 bp product
unamplified DNA as well as amplified DNA. However,
results from analysis of amplified DNA can be obtained Cvii uit.1

within a day to a week of the receipt of a sample of blood or I loot, 2Otbp I 1000 I 020$ I pA

other tissue, whereas the same results take one week to

“a’ I
several months when unamplified DNA is analyzed. The
five most commonly used methods for detecting sequence
variation for diagnosis of inherited diseases after PCR
amplification are the following: (a) endonuclease digestion F.-
of the PCR product followed by electrophoresis, (b) hybrid-
ization of the PCR product to an allele-specific oligonucle- AA AS SS SS SS AS SS
otide probe, (c) electrophoresis of the PCR product, (d) DNA
sequencing of the PCR product, and (e) polymorphism
analysis by either endonuclease digestion or hybridization
to oligonucleotide probes as described in a and b. For
purposes of this discussion, polymorphism analysis is con-
sidered a different method because the way in which the
data are interpreted for diagnostic purposes is quite dif-
ferent.
If a disease-producing gene has not yet been cloned, or
the disease-producing mutations present in the gene poo1
- 38lj3S
are not yet characterized, or if the number of mutations in
the gene pool is too large to permit efficient screening for all - 256, constant
alleles, diagnosis will be carried out by utilizing polymor- -cL180J
201-i.. IDA
phisms in linkage analysis. For diagnostic purposes, direct
detection of the mutation (methods a-cl) is more nearly
accurate, and thus is preferable to polymorphism analysis. - 88, constant
Errors due to meiotic recombination, to which polymor-
phism analysis is susceptible, are eliminated when detect-
ing the mutation directly. The method used to detect a
mutation directly depends on the type of mutation being
examined: single-nucleotide substitution that affects a re-
striction site, single-nucleotide substitution that does not
affect a restriction site, or complete or partial gene deletion. Fig. 2. (top) Cvn I map of a f3-globinregion725-basepair (bp)
amplifiedDNA productthat containsthe sequencealteredby the
EndonucleaseDigestionof PCR Product sicklecell mutation;(bottom) ethidiumbromidestain of Cv,, I-
Mutations that affect a restriction endonuclease recogni- digestedamplifiedDNAproduct from individuals with sicklecelltrait
(AS),normal/3-globin genes(AA),andsicklecellanemia(SS)
tion site can be detected without use of a DNA probe. The
(Top),the 5’ and 3’ endpointsof amplified product are determined by the two
sickle cell mutation is an example of this type of mutation. primers used in the amplification reaction. Arrows representCvn I sites; ‘the
This single nucleotide substitution (A-T), which causes an Cvn I site destroyed by the sickle cell mutation

1844 CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989

Hybridization with Allele-SpecificOligonucleotideProbes radish peroxidase (EC 1.11.1.7) have been shown to be as
When a point mutation does not affect a restriction sensitive and specific as probes radiolabeled with 32P (34).
endonuclease recognition site, it can be detected by use of
an oligonucleotide probe (3, 54). When used under strin- Agarose Gel Electrophoresisfor Detection of Deletions
gent conditions, these probes hybridize only to identical A deletion can be detected by agarose gel electrophoresis
sequence, and a single-nucleotide difference between probe of a PCR product without the need for either endonuclease
and genomic DNA is sufficient to destabilize the duplex digestion or probe hybridization. Either the deletion can be
molecule and prevent hybridization. Each oligonucleotide observed directly as an alteration in the size of an ampli-
probe is mutation-specific, and exact sequence alteration fication product, or its presence can be deduced by absence
must be known before a probe can be synthesized. of an amplification product, depending on whether the
The Tm (melting temperature) of a DNA duplex is sequences complementary to the primers are retained in
dependent on its nucleotide composition and length. The the deletion. Alternatively, an oligonucleotide probe that
longer the length of the sequence and the greater the G-C includes the sequence at the junction of a deletion can be
content, the higher the Tm. The specificity of an oligonu- utilized.
cleotide probe for identical sequence is achieved through When sequence complementary to the primers is present
being shorter than the probes used in Southern blotting, in DNA despite a deletion, the amplification reaction will
which are not sensitive to small differences in sequence.
result in a PCR product of different size from the normal
Stringency is increased by raising the temperature and (or) product. However, if the deletion spans a relatively large
decreasing the salt concentration of the post-hybridization
distance (-2 kb or more), different sets of primers may be
wash solution. Because of their ability to detect specific
necessary to amplifr the normal and deletion alleles. This
mutations, these probes are referred to as allele-specific
is the case when the distance between the primers used to
oligonucleotide probes.
For examination, the amplified DNA is immobilized on a detect a deletion is too large in the normal allele for the
nylon membrane by dot blotting under reduced pressure, polymerase to extend the total distance between them.
then hybridized against a labeled oligonucleotide probe If primers are available for a normal allele but the
(Figure 3). Positive and negative controls must be included sequence bordering one or both ends of a deletion is not
on each membrane to indicate whether the stringent salt known, primer sequences for the deletion cannot be syn-
and temperature conditions essential for probe specificity thesized. In this case, a deletion can be detected as the
have been met. A reverse strategy, in which the probe is absence of an amplification product when primers to the
immobilized on a ifiter and amplified DNA is supplied in normal allele are used (Figure 4). However, this detection
the hybridization solution, has been described and is re- procedure can only be used with samples from a homozy-
ferred to as “reverse dot blot” (55). gous affected individual in the case of an autosomal se-
The PCR produces abundant amounts of a specific DNA quence or from an affected male in the case of an X-linked
sequence available for hybridization. Therefore, the oligo- deletion. For detecting deletions that result in the absence
nucleotide probes used with amplified DNA can be less of a PCR product, a control set of primers complementary to
sensitive than those required for working with unamplifled another segment of the genome should be included in the
DNA. For amplified DNA, probes conjugated with horse- PCR reaction. Determination of the correct conditions for a
reaction that involves more than one set of primers, a
“multiplex” reaction, often requires trial and error. Alter-
Gene Amplification/Dot. Blot Hybridization ations in the temperature or length of time for primer
tCss.I N.rIICSI..3 sestyp. annealing, changes in the magnesium concentration
within the reaction buffer, and changes in the amount of
cix. #{149} .,A,I.
DNA added to the reaction can all affect the success of a
multiplex reaction, If conditions are not correct for multi-
.0. plexing, one might obtain amplification with one set of
primers but not the other, despite the fact that both sets are
$A#{216}A in the same reaction tube.
Multiplex PCR reactions have proven very useful for
$0,’. DNA diagnosis of Duchenne and Becker muscular dystro-
phy. In these disorders, 70% of cases are due to a dystrophin
gene rearrangement, usually partial deletion, which can be
detected by Southern blot analysis using cDNA probes. By
multiplexing with nine sets of PCR primers, chosen to
amplifr those regions of the gene that are susceptible to
Fig. 3. Autoradiogram
of a dot blotof amplified DNA hybridized deletion in Duchenne and Becker muscular dystrophy,
againstallele-specific
oligonucleotide
probes(ASO8)for prenatal Chamberlain et al. (37) showed that 90% of those deletions
diagnosisof /3-thalassemia detectable by Southern blot analysis are easily identified
Both parents inthe pedigree at leftcarrythe codon 39 mutation as demon- by the considerably quicker and easier PCR method (3; and
strated by hybridization of their amplified DNAs to the mutant ASO. Control personal communication, J. Chamberlain). In some cases it
samples, essential for monitoringthe specificityof the hybridization,are In the
3rd and 4th dots from the top and respectively represent amplified DNA from may be possible to detect unaffected carriers of deletions by
individualshomozygous for normal/3-globinalleles and homozygousfor the comparing intensities of the ethidium bromide-stained con-
codon 39 -thalassemia allele. Amplified DNA from this couple’s affectedchild trol PCR product relative to the PCR product that may be
hybridizes to mutant ASOonly;that from the fetus (broken outline) hybridizes
to boththe mutant and normal probes, demonstrating that the fetus is a carrier deleted in the heterozygote (J. Chamberlain, personal com-
of -thaIassemla munication).

CLINICALCHEMISTRY,Vol.35, No.9, 1989 1845


Ml 234 567
Fig. 4. Agarose gel electrophoresisafter amplificationdetects fi-
globingene deletion
Mother (lanes I & and father(lanes3 & 7) are both carriers of Hb Lepore,
a fusion protein that containsthe 5’ end of the 5-globingene and the3’end of
the p-globin gene. The sequence betweenthese two genes has been deleted
and neither intact 8- nor -globin genes are present on the Hb Lepore
chromosome.Lanes 1-4 show PCR productafteramplification of sequences
In the 5’ end of the p-globingene (upper arrow). In thisset of primers,
sequencescomplementarytothe3’primerarepresent in both the normal and
Lepore alleles,but onlynormalp-globin contains sequences complementary
to the 5’ primer. Thus, the Kb Lepore allele will not amplify with this set of
primers.PCR products are from(1) mother,whois a carrier ofHb Lepore; (
fetus of this couple, which is affected with Hb Lepore; ( father, who is a
carrier of Kb Lepore; and (4) a normal individual who is not a carrier of Hb
Lepore.Lane Mis a ,bX174 markerdigestedwithHae Ill. Lanes5-7show PCR
product after multiplexamplificationwiththe primerset for the 5’ end of the
normal globin gene (used inlanes 1-4) as wellas a primer set tothe 3’ end
of the frglobin gene, with which both normal /3-globinand Hb Lepore alleles
amplify (lower arrow). Products shown are from (5) the mother, (5) the fetus,
and (7) the father. While a diagnosis of a fetus affectedwith Hb Lepore Is
suspected from results in lane 2. in which there is no amplification ofnormal
globin genes, the diagnosis is more certain from results in lane 6, in whichthe
control set of primers indicates that DNA from the fetus was present and that
the conditions in the reaction tube supported amplification
Gene Sequencing
If the gene locus responsible for an inherited disorder is
known and has been at least partly characterized by
sequencing data, it may be relatively easy to identif’ the
mutation responsible for the disease state in an individual
by partly or totally sequencing the gene. Once a DNA
segment has been amplified, sequence data from the dou-
ble-stranded amplification product can be obtained within
a day by use of an oligonucleotide as a primer in a dideoxy
sequencing reaction (35, 43, 46, 56-58). Thus, mutant
alleles responsible for a single-gene disorder can be rapidly
characterized, especially in small genes such as the f3-
globin gene. For j3-globin, the entire gene of 1.8 kb can be
amplified with one set of primers and the gene sequence for
most of the gene obtained from the use of seven sequencing
primers (35). The gene-sequence approach may be chosen
for mutation identification when many different mutations
give rise to the same disorder (e.g., Lesch-Nyhan), making
impractical the use of allele-specific oligonucleotide probes
for diagnosis (59).
PolymorphismAnalysis
For most inherited disorders, the gene responsible for the
disease has not yet been identified. However, for some of
these unknown genes, restriction fragment length poly-
1846 CLINICAL CHEMISTRY, Vol.35, No. 9, 1989
morphisms (RFLPs) near the gene have been discovered,
such as for cystic fibrosis, Huntington’s disease, neurofibro-
matosis, and adult polycystic kidney disease. These RFLPs,
linked to the responsible gene, can be used in linkage
analysis to track the inheritance of the mutant and normal
genes within a family. In fact, linkage analysis has been
the most common type of DNA analysis used for prenatal
diagnosis of single-gene disorders since first used in DNA
testing for sickle cell anemia in 1979 (60). RFLPs are also
used for DNA diagnosis of some disorders in which the gene
has been isolated and characterized but for which the
number of mutations producing the disease among dif-
ferent families is so large that the use of direct mutation-
detection methods (as described earlier) is not feasible for
diagnosis in most cases. Such is the case in DNA testing for
hemophilia A and for those cases of Duchenne muscular
dystrophy in which the disease is not due to gene deletion.
An amplification product is typed for an RFLP by endo-
nuclease digestion, followed by agarose gel electrophoresis,
as described above. Although the methodology for detecting
an RFLP by use of the PCR technique is simple when
compared with the Southern blotting method, converting
the detection scheme for any particular RFLP from South-
ern blotting to the PCR technique may be very involved.
Many previously described RFLPs lie outside the probe
sequence with which they are detected on a Southern blot;
therefore, obtaining the sequence required for the genera-
tion of primers may require cloning and sequencing the
DNA that flanks the probe. This type of work has been
accomplished for the KM19 (Pst I) RFLP, which is linked to
the cystic fibrosis gene (39). A CS.7 (Hha I) RFLP linked to
the cystic fibrosis gene can also be typed by PCR analysis
(39). For RFLPs lying within a previously known sequence
(such as within the /3-globin gene and the Factor VIII gene),
the conversion to RFLP typing by PCR can be achieved
rapidly.
It is also possible to digest a multiplex reaction. In this
way, the identity of RFLP types associated with a particu-
lar chromosome (RFLP haplotype) can be determined after
a PCR reaction with multiple primer sets and endonuclease
digestion of the amplification product with multiple endo-
nucleases, all carried out simultaneously in the same
reaction tube. This has been achieved for five sites within
the f3-globin gene cluster (61).
If a polymorphism does not affect a restriction site, the
two alleles can also be distinguished through use of allele-
specific oligonucleotide probes, one for each allele.
The PCR technique is being used for prenatal diagnosis
and carrier testing of several inherited disorders. Although
it has not expanded the capabilities of DNA diagnosis, it
has improved DNA diagnosis significantly by allowing
diagnoses to be made more rapidly, and by eliminating the
need for radioactive probes in many instances.
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