Molecular Aspects of Myotonic Dystrophy Part 2 Flashcards
What challenges are associated with the molecular testing for myotonic dystrophy? How are these difficulties overcome?
For both DM1 and DM2 it is important to differentiate between repeats in the normal size range and expanded repeats.
Due to the large size of some expansions and the secondary structures formed by the repeat, conventional PCR can’t amplify larger alleles.
Differentiating between homozygosity for normal sized alleles of the same size and one normal allele with an undetectable expansion is also a problem.
A combination of techniques is used to overcome the problems.
Why can’t conventional PCR amplify some DM1 and DM2 expansions?
Due to the large size of some expansions and the secondary structures formed by the repeat, conventional PCR can’t amplify larger alleles.
What is usually the first line testing for DM1? Describe this method.
One of the first line techniques employed for all DM1 testing is a PCR designed to size the repeat tract. It identifies patients with two alleles in the normal size range and can detect small expansions.
By using a fluorescently tagged pair of primers that span the repeat the resulting sizes of the PCR products can be used to accurately calculate the number of CTG repeats.
PCR enhancers can be added to the mastermix to reduce the formation of secondary structures and improve the amplification of the region e.g. DMSO, Benzene.
One primer is fluorescently tagged to allow visualisation of the products by capillary electrophoresis.
The size of the PCR fragment is used to determine the number of repeats. For accurate sizing two controls of a known size are included with every batch. Controls are used with sizes close to the boundaries between normal, intermediate and the affected ranges. For example, one control with 5+51 repeats and another with 13+36. A blank negative control is also used to rule out the presence of contamination.
Can detect expansions between 70 and 80 repeats. Is useful for detection of patients with 2 alleles in the normal size range.
Advantages:
- Cheap and easy to set up
- Quick method to rule out DM patients with 2 normal alleles of different sizes
- Accurate sizing of normal alleles
Disadvantages:
- SNP beneath a primer site could give false negative result
- Not able to detect expansion above approximately 80 repeats
- Can’t differentiate between homozygous alleles and one normal allele with large undetectable expansion
What is the function of PCR enhancers in the first line PCR for DM1 testing?
PCR enhancers can be added to the mastermix to reduce the formation of secondary structures and improve the amplification of the region e.g. DMSO, Benzene.
In DM1 repeat sizing PCR how do we ensure that the sizing around the boundaries is accurate?
The size of the PCR fragment is used to determine the number of repeats.
For accurate sizing two controls of a known size are included with every batch.
Controls are used with sizes close to the boundaries between normal, intermediate and the affected ranges. For example, one control with 5+51 repeats and another with 13+36.
A blank negative control is also used to rule out the presence of contamination.
Why are larger alleles often represented by a hedgehog type pattern on repeat sizing PCR?
Because the smaller allele is preferentially amplified and so you get a strong signal for the smaller allele and a hedgehog like pattern for the larger allele.
What are the advantages and disadvantages of repeat sizing PCR for DM testing?
Advantages:
- Cheap and easy to set up
- Quick method to rule out DM patients with 2 normal alleles of different sizes
- Accurate sizing of normal alleles
Disadvantages:
- SNP beneath a primer site could give false negative result
- Not able to detect expansion above approximately 80 repeats
- Can’t differentiate between homozygous alleles and one normal allele with large undetectable expansion
What additional DM testing method is utilised to overcome the shortcomings of the repeat sizing PCR technique?
Triplet primed PCR.
What is TP-PCR? How can it be used in combination with repeat sizing PCR in DM1 testing?
TP-PCR is designed to detect the presence of an expansion, although the technique is unable to size the expansion.
The combination of TP-PCR and repeat sizing PCR can give a reportable result in nearly all patients.
TP-PCR can detect the presence of large expansions of the repeat tract.
TP-PCR is usually run in parallel with the sizing PCR to save time. The combination of the two PCRs allows for the detection of large expansions and can confirm homozygosity for a normal sized allele.
By using the two methods the risk of a false result is reduced.
Positive and negative controls are run in addition to a blank negative.
Describe the primer system used for TP-PCR in myotonic dystrophy testing.
TP-PCR utilises a 3 primer system.
1) . The forward primer binds to a sequence upstream of the repeat. The forward primer is fluorescently labelled for capillary electrophoresis.
2) . The second primer can be described as the reverse primer. This primer is made up of 2 halves. The 3’ end is complimentary to the repeat so primes at multiple locations. The 5’ end is non-specific.
The reverse primer is at a limited concentration so that it is exhausted after the first few PCR cycles. This prevents priming from PCR products that would lead to a decreasing length of products in the final pool. Limiting the levels of reverse primer means that a representative output of product is maintained.
3). To allow further amplification of the products from the forward and reverse primers a tail primer is used that is specific to the 5’ end of the reverse primer.
The final pool of products represents the whole repeat giving a distinctive trace after capillary electrophoresis.
Describe the reverse primer used in TP-PCR.
This primer is made up of 2 halves. The 3’ end is complimentary to the repeat so primes at multiple locations. The 5’ end is non-specific.
Which end of the TP-PCR reverse primer is non-specific?
The 5’ end is non-specific.
Which end of the TP-PCR reverse primer is complimentary to the repeat sequence?
The 3’ end is complimentary to the repeat so primes at multiple locations.
In TP-PCR why is the reverse primer at a limited concentration?
The reverse primer is at a limited concentration so that it is exhausted after the first few PCR cycles. This prevents priming from PCR products that would lead to a decreasing length of products in the final pool.
What is the function of the tail primer in TP-PCR?
The tail primer is specific to the 5’ end of the reverse primer.
The tail primer allows further amplification of the products from the forward and reverse primers.
The final pool of products represents the whole repeat giving a distinctive trace after capillary electrophoresis.
Without the tail primer there would be insufficient amplification to view the products.
