Blood samples

• Karyotype: 5-10ml blood in lithium-heparin blood tube
• DNA: 5-10ml blood in EDTA blood tube

Skin biopsy

• Punch biopsy (3-5mm) placed in transport medium for fibroblast culture
• Can be used for karyotype, biochemical assays and DNA analysis

The lymphocytes from a 10ml blood sample yield approximately 300ug of DNA, sufficient for multiple DNA analyses.  DNA can be stored frozen for very many years.

Polymerase Chain Reaction (PCR)

• Used to amplify a DNA sequence of interest (see figure)
• Two oligonucleotide primers are used which are complimentary to the DNA sequence of interest
• Firstly, the double stranded DNA is denatured by heat into single-stranded DNA fragments
• The reaction is then cooled to allow annealing (binding) of primers to the target DNA sequence
• Heat-stable Taq1 DNA polymerase then extends the primers along the template target sequence
• After one cycle there are 2 copies of double-stranded DNA, after two cycles there are 4 copies and so on
• Typically a PCR reaction involves 25-30 cycles, hence allowing millions of DNA amplifications

DNA Sequencing

• Di-deoxy sequencing allows identification of the exact nucleotide sequence of DNA
• The region of interest is amplified by a PCR reaction (see above). This builds a new DNA chain complementary to the region of interest
• The reaction is carried out in 1 tube, containing all four nucleo-tides and all 4 di-deoxy nucleotides (adenine, cytosine, guanine, thymine.
• The di-deoxy nucleotides are incorporated into the growing chain and where incorporated stop the addition of any further nucleotides
• The fragments are electrophoresed and the DNA sequence is determined by fluorescence
• DNA sequencing can be used to identify point mutations, small deletions and insertions.

Southern Blotting

• DNA is cut into fragments using a restriction enzyme that recognises a specific DNA sequence
• The fragments are separated according to size by gel electro-phoresis and transferred (blotted) to filter paper
• A probe is then used to bind to its complimentary sequence on the blot and can be identified by autoradiography
• Southern Blotting requires relatively large amounts of good quality DNA and is time-consuming

Multiplex Ligation-dependent Probe Amplification (MLPA)

• This is a relatively new, high resolution method to detect copy number variation in genomic sequences eg. large gene deletions and duplications
• Often gene analysis now involves DNA sequencing and MLPA to identify different types of mutation
• MLPA can also be used instead of FISH for the analysis of telomeres

Karyotyping (chromosome analysis)

• Samples are cultured
• Cell division is then stopped in metaphase
• Chromosomes stained with Giesma i.e. G-banding
• Alternating light (euchromatin) and dark (heterochromatin) bands are characteristic for each chromosome pair using light microscopy
• The resolution of G-banding is approximately 5Mb

Fluorescent In Situ Hybridisation (FISH)

• Technique used to detect the presence of specific DNA se-quences on chromosomes
• Uses fluorescent probes and light microscopy

Telomeric FISH

• FISH analysis using probes specific for telomeres (chromosome ends).
• Improves sensitivity of microdeletions or other cryptic rear-rangements as most telomeres are G-band-negative

Haematological malignancies and solid tumours

• Haematological neoplasms and paediatric solid tumours can be characterized by acquired genetic rearrangements detectable using karyotype analysis
• Used to provide information about diagnosis eg Philadelphia chromosome (9;22 translocation) in CML, for prognosis and remission

Technology using molecular DNA analysis has recently been developed to determine genomic copy number.

Array-CGH (comparative genomic hybridisdation)

• Patient and control DNA are given different fluorescent labels and hybridised to arrays containing thousands of genomic clones
• The fluorescent signals are compared to determine whether there is a change in genomic copy number (deletions or duplications) of patient DNA relative to control DNA
• The resolution is much greater than routine karyotyping and FISH
• However, it cannot detect apparently balanced rearrangements
• A number of variants have been identified with as yet unknown significance.
• Therefore, this technology is still being used with caution in determining results.
• In the future, this technology is likely to significantly increase the ability to diagnose chromosome abnormalities as a cause of mental retardation and congenital defects.

Quantitative fluorescence polymerase chain reaction (QF-PCR)

• QF-PCR is used to test for gene dosage, ie the number of copies of a given gene present in a sample.
• Examples of its use includes testing for aneuploidy of whole chromosomes eg 13, 18, 21, X, Y in a CVS or amniotic fluid
• Markers of DNA from the sample are amplified, labelled with fluorescent tags and measured by electrophoresis.

Amniocentesis

• Under ultrasound guidance, needle inserted transadominally through uterine wall for aspiration of amniotic fluid
• From 15 weeks gestation
• Miscarriage risk 1% above normal population risk
• Can be used for fetal karyotype, biochemical assays and DNA analysis

Chorionic villus sampling (CVS)

• Under ultrasound guidance, needle inserted transadominally through uterine wall for biopsy of chorionic villi
• From 11 weeks gestation
• Miscarriage risk 1% above normal population risk
• Can be used for fetal karyotype, biochemical assays and DNA analysis

PGD is a form of genetic diagnosis performed prior to implantation.

• The patient’s oocytes are fertilised in vitro and, at the 8 cell stage of embryogenesis, a single cell is removed and analysed for the genetic condition in question.
• The diagnosis itself can be carried out using several techniques, eg FISH and PCR