Fragment Sizing Strategies

Question 1

What considerations should be made before undertaking a fragment sizing experiment?

Answer 1

If native DNA is to beused as the imput material then fragmentation is required as the DNA will be either too large to pass through the gel matrix or supercoiled, and pass through at a rate that is disproportional to its size.

Alternatively, PCR products do not rely on this preparation howeevr allele drop out and preferential amplification may hinder the estimation of fragment sizes.

Similarly DNA fragmentation of native DNA needs to run to completion, otherwise partial fragments will hinder size estimation.

Question 2

Name 6 different methods used to size separation DNA based on eletrophoresis.

Answer 2

1. Agarose gel electrophoresis:
2. Polyacrylamide gel electrophoresis (PAGE)
3. Pulse field gel electrophoresis
4. Capillary electrophoresis
5. Nanowire structures
6. Bioanalyzer Alilent)

Question 3

Describe Agarose gel electrophoresis

Answer 3

1. DNA is mixed with a loading dye and loaded into wells.
2. The DNA is negatively charged due to the phosphate backbone and will run to the positive electrode.
3. The speed of migration is dependent on the current and percentage gel. Larger fragments will migrate more slowly.
4. The agarose percentage can be altered to allow varying degrees of resolution.
5. A fluorescent substrate can be added to the agarose prior to gel casting or after the gel has been ran by soaking the gel in a buffer containing the substrate.
6. It is necessary to run an appropriate size marker (e.g 1kb ladder) alongside the samples in order to estimate the size of the fragments.
7. Agarose gel electrophoresis can resolve fragments ~50bp to 30kb in length.

Question 4

Describe Polyacrylamide gel electrophoresis (PAGE)

Answer 4

1. More sensitive than agarose gels and can resolve fragments <50bp in length
2. DNA molecules can be separated that differ as little as 0.2% (e.g. 1bp in 500bp). Formally used for the evaluation of SMA and heteroduplex analysis.
3. Staining methods include silver staining.
4. A molecular marker and controls should be used to provide a size comparison.

Question 5

Describe Pulse field gel electrophoresis

Answer 5

1. Used for the resolution of large DNA fragments (~20kb to several Mb).
2. The relative orientation of the gel and the electric field is altered. The migration of DNA molecules is shortened, as they re-orientate themselves towards the positive electrode.
3. The larger fragments take longer to migrate, thus the fragments form tighter clusters of DNA fragments that can be visualised as separate bands.

Question 6

Describe Capillary electrophoresis

Answer 6

1. Used in combination with fluorescent tags, this process allows the sizing of DNA fragments to within 1bp of each other.
2. The DNA fragments are denatured and placed on a machine where the single stranded DNA migrates through the charged capillary containing polyacrylamide gel.
3. The rate of migration is dependent on the size of the fragment and requires an internal size standard to be run for each sample.
4. Amplified fragments can be mixed, providing they are of different size and further multiplexing can be undertaken through the application of different fluorophores.
5. Applications for this include MLPA, genotyping such as QF-PCR for the analysis of aneuploidies and microsatellite analysis of tumour samples (HNPCC / Lynch syndrome) and sanger sequencing.

Question 7

Describe Nanowire structures

Answer 7

1. 3D nanowire structures embedded in microchannels can be used for ultra fast separation of small biomolecules such as DNA (50-1000bp) or RNA (100-1000bases).
2. The DNA or RNA mixture is stained with a dye. The electrophoretic mobility difference of the biomolecules is assessed as a function of molecular size in the 3D nanowire structure.

Question 8

Describe Bioanalyzer analysis

Answer 8

1. The Agilent Bioanalyzer is a nanofluidics device that preforms size fractionation and quantification of small samples of DNA, RNA, or protein on a single platform, providing high quality digital data.
2. Uses both electrophoresis and flow cytometry and can use small quantities of sample (1-4µL).
3. Up to 12 samples can be run on a chip. Each DNA chip contains an interconnected set of microchannels that is used for separation of nucleic acid fragments based on their size as they are driven through it electrophoretically.
4. Typically used to assess the quality and fragmentation of DNA prior to NGS library prep and to check the quality and quantity of NGS library preps.

Question 9

Describe Bioanalyzer analysis as used for RNA degredation.

Answer 9

Bioanalyser can be used to assess RNA degradation by calculating the RIN (RNA integrity number) of RNA samples. As RNA degrades, there is a decrease in the ratio of ribosomal bands18S and 28S and an increase in the base line signal between the two peaks.

Question 10

Name 3 different methods used to size separation DNA based on PCR.

Answer 10

1. Long range PCR
   
   • Barnes. (1994) Proc. Natl. Acad. Sci. USA 91, 2216–2220
2. Fluorescent PCR
3. Triplet-primed PCR
   
   • Warner et al., 1996 J Med Genet. 33(12):1022-1026

Question 11

Describe long-range PCR

Answer 11

1. Normal PCR can be modified to amplify larger fragments (usually used to describe amplification of fragments > 5 kb)
2. Additives to overcome problems with high GC content: betaine equalises AT and GC contributions to strand pairing, DMSO destabilises secondary structure and weakens base pairing, deaza-dGTP is base analogue to dGTP and also reduces secondary structure
3. Often have mix of polymerases
   
   • Taq is a highly processive polymerase but lacks 3’ to 5’ exonuclease so introduces errors (~1 error per 1 kb)
   • Pfu or Pwo are proofreading enzymes that can correct errors introduced by Taq

Question 12

Give examples of long-range PCR used in diagnostic setting

Answer 12

1. Large common IKBKG deletion in Incontinentia Pigmenti
2. Haemophilia A intron 22 inversion and involves the amplification of fragments of 10, 11 and 12 kb
3. Provide templates for targeted NGS - especially where there is a pseudogene e.g. Polycystic Kidney Disease.

Question 13

Describe Fluorescent PCR

Answer 13

1. PCR with one primer with a fluorescent tag
2. Products are analysed by capillary electrophoresis
3. able to resolve products 1 bp apart
4. limited by size of fragment able to be amplified by PCR (~5 kb - depends on template, conditions, etc - see long PCR strategies)
5. Preferential amplification of smaller fragments means that large alleles may not be detected when present with smaller ones.

Question 14

What is Triplet-primed PCR used for?

Answer 14

Used to detect repeat expansions which are too big to amplify by conventional PCR

Question 15

Describe Triplet-primed PCR

Answer 15

1. A specific flanking fluorescently labelled primer (P1) used in combination with a pair of primers (P3, P4) amplifying from multiple priming sites within the repeat.
2. The 3’ end of the reverse primer (P4) is complementary to the repeat tract and binds at multiple sites resulting in a mixture of products.
3. The 5’ of this P4 primer complementary to the P3 primer.
4. A 10:1 molar ratio of P3 to P4 ensures that the P4 primer is exhausted in the early amplification cycles.
5. This reduces the priming of P4 to PCR products generated in these initial cycles, thus minimizing the shortening of the average PCR product size.
6. Primer P3 amplifies from the end of products of previous amplification rounds

Question 16

The following image shows Data for TP PCR only for three FRDA individuals. Describe the data trace.

Answer 16

1. TP PCR representative electropherograms, aligned by size (bp). The size standard peaks are shown by vertical segments.
2. The TP PCR signal consists of a ladder with 3bp periodicity, corresponding to the GAA repeat.
3. Both normal and expanded alleles give peaks. The ladder peaks diminishes gradually with increasing product size.
4. A) Absence of expansion. Amplification of a low number of repeats, below the threshold for expansion (~250bp).
5. B and C) Presence of expansion. The ladders extend along the electrophoretic run, beyond the threshold for the expansion

Question 17

Do all TP-PCR assays require 3 primers?

Answer 17

TP-PCR has been described for HD testing similar to the method above but only uses only two primers known as ‘chimeric PCR’

(Jama et al., 2013 J Mol Diag 15 (2): 255-262)

Question 18

Describe the 2 primer TP-PCR HD assay

Answer 18

1. Forward primer is locus specific
2. Referred to as chimeric PCR as the reverse primer has two or more annealing sites.
3. The 5’ end is specific to the region of the HTT gene following the CAG repeat and the 3’ end is complementary to 5 CAG repeats (chimeric portion).
4. The whole complementary primer anneals at a high temperature, generating a prominent peak specific to the full CAG repeat for each allele, whereas at a low annealing temperature, the chimeric portion of the primer anneals to generate less prominent peaks that form the CAG stuttering pattern

Question 19

What are the advantages of utilising TP-PCR?

Answer 19

1. Can greatly reduce the number of Southern blots required which are expensive to time consuming. If a normal patient is apparently homozygous in standard PCR then it’s not possible to know if the second allele is the fully expanded. TP-PCR can rule-out the presence of an expansion, negating the need for SB.
2. The periodicity of the peaks will indicate the number of repeats for a given repeat disorder, however TP-PCR shouldn;t be used for accurate sizing.

Question 20

Name some disdvantages of TP-PCR?

Answer 20

False negatives have been reported in DM1 and DM2 due to;

1. SNPs in the P1 binding site - therefore bidirectional TP-PCR is required.
2. Repeat interruptions disrupting the TP-PCR reaction.

Question 21

What is Southern blotting?

Answer 21

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples.

Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization

Question 22

What is the primary use for Southern Blotting in diagnostics?

Answer 22

1. Used for detection of large fragments not amplifiable by PCR
2. 3. Can provide information about methylation status if methylation-sensitive enzyme are used (useful for FraX).

Question 23

There are 8 key stages to performing a Southern Blot, what are they?

Answer 23

1. Isolate Genomic DNA
2. Restriction digest
3. Gel electrophoresis
4. Transfer
5. Labelling probe
6. Hybridisation
7. Washing
8. Detection

Question 24

Describe the Isolation of Genomic DNA stage of Southern Blotting.

Answer 24

No PCR amplification required, need large amounts of DNA ~10 mg.

Controls are required to identify natural variation (e.g. Alu insertion in DM1 blots) or premutations in some syndromes (e.g. FraX)

Question 25

Describe the restriction digest stage of Southern Blotting.

Answer 25

1. required to isolate fragment of interest (can be double digest with methylation-sensitive enzyme, sequence changes can alter restriction sites giving atypical results).
2. Different restriction fragments can be used to reduce the likelihood of RFLPs causing ambiguous results. Furthermore different enzymes will result in different fragment lengths and allow varying degrees of resolution depending on the size to be estimated.

Question 26

Describe the Gel electrophoresis stage of Southern Blotting.

Answer 26

Digested DNA is resolved by electrophoresis (can use pulse-field gel electrophoresis for very large fragments)

After electrophoresis DNA in gel is denatured to make it single stranded (can also depurinate to fragment DNA as smaller fragments transfer more efficiently)

Question 27

Describe the ‘transfer’ stage of Southern Blotting.

Answer 27

DNA is transferred from the gel to a membrane (positively charged nylon or nitrocellulose) usually by capillary action where an absorbent material (paper towels) soaks up buffer through the gel and membrane taking the DNA with it (overnight).

Alternative blotting methods: Electroblotting & Vacuum blotting (30 mins)

DNA is then fixed to membrane by baking, sodium hydroxide or UV light. Nylon membranes cannot be damaged by handling so a single blot can be re-hybridised up to 10 times.

Question 28

Describe the ‘Labelling ‘ stage of Southern Blotting.

Answer 28

A probe is designed to the target sequence and used as a template to generate a labelled fragment.

The probe itseld can be created by PCR or digested from plasmid.

Labelling is achieved by random priming, nick translation or end labelling with [32P]-dCTP.

Non-radioactive methods more currently used using a chemiluminescent probe e.g. digoxigenin (DIG)

Question 29

Describe the ‘Hybridisation ‘ stage of Southern Blotting.

Answer 29

Hybridisation of the single stranded probe is usually carried out overnight for maximum efficiency

Pre-hybridisation is carried out to block unused DNA binding sites on the membrane surface (e.g. using salmon sperm DNA, BSA or dried milk)

Optimal stringency for hybridisation is achieved by the appropriate temperature, pH and salt concentration and detergent (e.g. SDS).

10-100 fold increased sensitivity can be obtained by including an inert polymer such as 10% dextran sulphate or 8% polyethylene glycol 6000

Question 30

Describe the ‘Washing ‘ stage of Southern Blotting.

Answer 30

After hybridisation washing removes unbound probe and hopefully any probe bound to non-specific sequences

The stringency of washes can be gradually increased by reducing the salt concentration in the wash buffers to achieve the appropriate signal (can be assessed with Geiger counter after each wash when using radioactivity)

Denaturants such as SDS and sodium pyrophosphate can increase the stringency of the washes

Question 31

Describe the ‘detection ‘ stage of Southern Blotting.

Answer 31

Radiolabelled blots can be visualised by autoradiography (low temperature and intensifying screens can increase the signal)

Question 32

What Alternative labelling systems are available for blotting other than radioactivity?

Answer 32

Chemiluminescence utilising DIG

1. DIG-labelled nucleotides may be incorporated, at a defined density, into nucleic acid probes by DNA polymerases or terminal transferase
2. DIG label may be added by random primed labelling, nick translation, PCR, 3’-end labelling/tailing, or in vitro transcription
3. Hybridized DIG-labelled probes may be detected with high affinity anti-digoxigenin (anti-DIG) antibodies
4. The anti-DIG antibodies are conjugated to peroxidase which emits low-intensity light in the presence of substrate.

Question 33

What are the advantages of Southern Blotting?

Answer 33

1. Can be used for very large expansion and non-STR disorders e.g. FSHD.
2. Minimise flase-negatives associated with PCR based methods (SNPs under primer, interspersions)
3. Potential to detect mosaicism for multiple expanded alleles if present.
4. Can detect methylation status by using methylation specific enyzymes if necessary e.g. Frax

Question 34

What are the disadvantages of Southern Blotting?

Answer 34

1. Labour intensive.
2. Not optimised for high throughput testing.
3. Expensive per sample costs if not batched.
4. Difficult interpetation.
5. Variable quality, highly dependant on skill of operator.
6. Large input DNA requirements (~5ug per enzyme per sample) thus not practical for all applications

Question 35

What alternative solutions are available to help labs reduce the number of Southern Blots peformed?

Answer 35

Several PCR based kits have become available that solve the problem of large expansions and methylation status detection without the requirement for blotting

e.g. Asuragen AmplideX kit: Reduces the need for Southern blotting to 2% or less of all samples

Chen et al., 2011, Genetics in Medicine 13(6): 528-538

Question 36

Describe the Asuragen AmplideX kit .

Answer 36

Provides a high throughput PCR and capillary electrophoresis analysis workflow that can accurately resolve;

1. sample zygosity
2. identifies AGG spacer elements
3. differentiate the full range of mutation alleles

Question 37

How does the Asuragen AmplideX kit work?

Answer 37

1. All samples are amplified by a TP-PCR method and only expanded alleles need assessment for methylation status by methylation-sensitive digest analysis.
2. HpaII digested DNA and non-digested DNA are amplified in 2 separate reactions with different fluorescent labels (FAM and HEX).
3. Products from both reactions are combined together for capillary electrophoresis and percentage of methylation is calculated by ratio of peaks for digested and undigested DNA.

Question 38

FSHD is special kind of expansion disorder because it’s repeat unit is very large. What methods have been developed to replace Southern Blotting for FSHD?

Answer 38

Molecular combing enables the identification of large DNA rearrangements (4kb to 1Mb) by direct visualization and analysis of single DNA molecules.

It involves dipping chemically modified glass coverslips into a DNA solution.

The coverslip is then pulled out at a slow and constant speed, DNA molecules become irreversibly attached and stretched on the coverslip.

Labelled DNA probes can be hybridized on combed DNA and detected by fluorescence microscopy.

This method can be used for Facio-scapulo-humeralmusculardystrophy (FSHD) as an alternative and improvement to Southern blotting due to its analytical ability to directly visualise the contraction of the D4ZA repeat array on chromosome 4.

Question 39

How can whole genome sequencing technology be utilised as a fragment sizing tool?

Answer 39

1. Bioinformatic tools can utilise PCR-free WGS data to identify repeat expansions even if the expansion is larger than the read length.
2. ExpansionHunter is the most recent algorithm for this and utilised WGS to accuraely size C9orf72 expansions as a proof of concept.
3. ExpansionHunter correctly classified 99.5% (212/213) of the expanded samples and all (2,788/2,788) of the wild type samples.