Lecture Nine

Multiplex PCR

Locus A Locus E
Locus B Locus F
Locus C Locus G
Locus D Locus H
 Locus A Locus B

Allelic size 220-390bps 370-500bps

range

Genomic DNA
A ? ? B

252bps 372bps 384bps 484bps

Mobility modifying non-nucleotide linkers introduced with the
Identifiler™ kit (Applied Biosystems)

• The mobility modifier is composed of hexaethyleneoxide (HEO) that

imparts a shift of approximately 2.5 nucleotides with each additional HEO
unit.

• This non-nucleotide linker is synthesized in the 5′-end of the PCR primer

so that when the PCR product is created it contains these extra molecules
on one end.

• By incorporating non-nucleotide linkers, mobilities for amplified alleles

from one member of a pair of closely spaced STR loci can be shifted
relative to the other.

• Thus, overlapping size ranges can be prevented.

Mobility modifying non-nucleotide linkers introduced with the
Identifiler™ kit (Applied Biosystems)
 Gender identification using Amelogenin

• Gender-typing is commonly performed in conjunction with STR analysis.

• Amelogenin is a gene that codes for proteins found in tooth enamel.

• These primers flank a 6 bp deletion within intron 1 of the amelogenin

gene on the X homologue.
 Gender identification using Amelogenin

• Other regions of the amelogenin gene have size differences between the X
and Y homologues and may be exploited for sex-typing purposes.

• For example, a research group (1994) used a single set of primers that
generated a 977 bp product for the X chromosome and a 788 bp fragment
for the Y chromosome.

• In this case, a 189 bp deletion in the Y relative to the X chromosome was

used to differentiate the two chromosomes.
 Pitfalls; Gender identification using Amelogenin

• The results are not full-proof.

• A rare deletion of the amelogenin gene on the Y chromosome can cause

the Y chromosome amplicon to be absent.

• In such a case, a male sample would falsely appear as a female.

• It appears that this deletion of the Y chromosome amelogenin region is

more common in Indian populations than those of European or African
origins.
 Interpretation of STR profiles; Internal standards

• Internal-lane size standard is used to measure the size of PCR products

(alleles).

• Contains DNA-fragments of known lengths that are labelled with a

fluorescent dye, and the fragments are detected along with the amplified
PCR products during capillary electrophoresis.

• As internal-lane size standard is analysed along with each PCR, any

differences between runs that could affect the migration rates during
electrophoresis, such as temperature, do not impact on analysis.

• The software generates a size calling curve from the internal-lane size
standards – the data point of the unknown fragments are compared to the
size calling curve.
Interpretation of STR profiles; Internal standards
 Interpretation of STR profiles; Allelic ladder

• Because some STR alleles differ by only one base pair, the use of allelic
ladders that contain all the common alleles at each locus has been
adopted by the forensic community to ensure accurate profiling.

• Unlike the internal-lane size standards the allelic ladders cannot be

analysed in the same injection as the samples but are run periodically
during the analysis of a batch of samples.
 Allelic ladders

• An allelic ladder is an artificial mixture of the common alleles present in

the human population for a particular STR marker.

• They are generated with the same primers as tested samples and thus
provide a reference DNA size for each allele included in the ladder.

• Allelic ladders have been shown to be important for accurate genotype

determinations.

• Allelic ladders are constructed by combining genomic DNA or locus-

specific PCR products from multiple individuals in a population.
 Allelic ladders
• For example, to produce a ladder containing five alleles with 6, 7, 8, 9, and 10
repeats, individual samples with genotypes of (6,8), (7,10) and (9,9) could be
combined.

• Alternatively, the combination of genotypes could be (6,9), (7,8), and (10,10)

or (6,6), (7,7), (8,8), (9,9) and (10,10).

• Additional quantities of the same allelic ladder (second- and third-generation

ladders) may be produced by simply diluting the original ladder 1/1000–1/1
000 000 parts with deionized water and then re-amplifying it using the same
PCR primers .
 Detection of STR polymorphisms; Silver staining

• Some STR alleles differ by only one base pair.

AATG AATG AATG AATG AATG AATG AATG AAT AATG AATG TH01; 9.3
AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG TH01; 10

• Early systems detected the PCR products after electrophoresis on

polyacrylamide slab-gels using silver staining.

• Gel electrophoresis of the PCR products through denaturing polyacrylamide

gels can be used to separate DNA molecules between 20 and 500
nucleotides long with single base pair resolution.

• More sensitive than EtBr staining and can be used with denatured (single
stranded) DNA.
 Detection of STR polymorphisms

• Involves reduction and deposition of silver cations around the

DNA.

• This limited the number of loci that could be incorporated into

the multiplexes because the allelic size ranges of the different
loci could not overlap.

• To overcome this limitation, fluorescence labelling of PCR

products followed by multicolour detection has been adopted
by the forensic community.
Detection of STR polymorphisms; capillary electrophoresis (CE)

• The electrophoresis platforms have evolved from systems based on slab-

gels to capillary electrophoresis (CE) that use a narrow glass tube filled
with an entangled polymer solution to separate the DNA molecules.

• Applied Biosystems provide the most commonly used capillary

electrophoresis systems and all these have multicolour detection capacity.

• The ABI PRISM®310 Genetic Analyzer that has a single capillary and
analyses up to 48 samples per day.

• The ABI PRISM®3100 and Applied Biosystems 3130xl Genetic Analyzers,

which have 16 capillaries and can analyse over 1000 samples per day.

• The ABI PRISM®3700 and Applied Biosystems 3730xl Genetic Analyzers,

which can have up to 96 capillaries that can analyse over 4000 samples per
day.
 Detection of STR polymorphisms; capillary electrophoresis (CE)
• Before electrophoresis, the PCR sample is prepared by mixing
approximately 1μl of the reaction with 10–20μl of deionized formamide.

• The formamide denatures the DNA,

• Heating the samples to 95◦C is routinely done to ensure that the PCR
products are single stranded.

• The internal-lane size standard is also added at this point.

• Samples are transferred into the capillary using electro-kinetic injection- a

voltage is applied and charged molecules, including the amplified DNA
fragments and the internal-lane size standards, migrate into the capillary.

• After injection, a constant voltage is applied across the capillary and the
PCR products migrate towards the positively charged anode, travelling
through the polymer, which fills the capillary and acts as the sieving matrix.
Detection of STR polymorphisms; capillary electrophoresis (CE)

• Urea and 2-pyrrolidinone in the gel polymer and a temperature of 60◦C

help to prevent the formation of any secondary structure during
electrophoresis.

• Through-out the period of electrophoresis, an argon ion laser is shone

through a small glass window in the capillary.

• As PCR products labelled with fluorescent dyes travel past the window
they are excited by the laser, emit fluorescence that is detected by a
charged coupled device camera (CCD), and then are recorded by collection
software.

• The electrophoresis of a sample takes up to 30 minutes after which the

polymer in the capillary is replaced with fresh polymer and the next
sample can be analysed.
 Internal size
Capillary electrophoresis (CE)
standards

DNA, Formamide,
1 μl 10–20 μl

Heat, 95◦C Gel Matrix:

Polyacrylamide Constant voltage
Electrokinetic
Urea 60◦C
injection 2-pyrrolidinone
Cathode

Anode