16. Molecular genetic methods and applications in human genetics III Flashcards
Applications of genetic tests
1) Diagnostic/predictive
- Direct detection of mutation, confirm/excude mutation or qualify prognosis
2) Population wide screening
3) Prenatal diagnosis
Prenatal diagnosis
Invasive
- CV=chorionic villus sampling (10-12 wks gestation)
- Amniocentesis (14-16 wks gestation)
Non-invasive
- Maternal serum screening at 16 wks (AFP)
- US
- Study of fetal cells from maternal blood
Preimplantation diagnosis
Non-invasive studies to test Down syndrome
1) US - nuchal translucency increased + nasal bone absent
2) Maternal age
3) Maternal serum markers - hCG increased
Maternal serum markers
- PAPP-A (pregnancy associated plasma protein A
- AFP (alphafetoprotein)
- Estriol (UE3)
- Inhibin-A (inhibin)
- hCG (human chorion gonadotrophin)
Microsatellites
Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within the alleles of an individual - kind of a “fingerprint”.
- PCR of chromosome specific markers
- Capillary electrophoresis
PrenaTest
Non-invasive prenatal testing
- Can identify 7 chromosomal disorders
Comparative genome hybridization (CGH)
Whole genome of patient and control as samples
- Nick translation
- Fluorescent dyes - red for patient, green for control
- Chromosomes are fixed in metaphase + hybridized to slide
Results:
- Where patient and control are equally hybridized - rate of fluorescent signal is also equal (=1 - yellow)
- More intense patient signal - genome has extra copies
- Less intense patient signal - genome has a deficit
CGH advantages compared to karyotyping and FISH + disadvantage
1) Advantages over karyotyping
- Easily noticable differences
- Higher resolution (100 kb vs 5-10Mb)
2) Advantages over FISH
- Can examine more than only specific, pre-selected regions
3) Disadvantage
- Contamination (in tumor samples are also healthy cells)
Microarray technology
High resolution nucleotide chain hybridization
1) Nucleotide probes bound to solid surface (glass chip, bead)
2) Labelled samples are hybridized to probes
3) Measure signal intensity
Microarray applications
- aCGH/CNV/SNP
- Gene expression
- microRNA
- Methylation array
Clinical use of aCGH
- Disease focused arrays (DMD array)
- Hematological and cancer arrays
- Prenatal arrays
- Preimplantation arrays
Gene expression microarray
- Mainly in research
- Compare mRNA expression in tested samples
- Complex bioinformatical analysis (GSEA)
Gene expression microarray applications
- Microbiology-pathogen and host interaction studies
- Drug development, drug efficiency testing
- Biomarker studies (tumor diagnostic)
MammaPrint
Check breast cancer - risk of recurrence within 10 years after diagnosis (early stage breast cancer)
- Low risk vs high risk
- Expression pattern of 70 genes
- Low risk: don’t need chemo after surgery
microRNA microarray
microRNA profile is distinct in healthy and cancer tissues, or between tumors
Methylation detection by PCR
Bisulfite PCR, MS-PCR (methylation sensitive)
- Treatment of DNA with bisulfite converts cytosine residues to uracil in CpG sites - but leaves 5-methylcytosine residues unaffected
- During PCR unmethylated cytosines are displayed as thymines in the resulting amplified sequence
- Can be verified by sequencing
Methylation microarray
- Used to test DNA methylation
- Used for epigenetic studies
Huntington disease detection
PCR
- Detection of a length polymorphism (VNTR)
- CAG repeats
- Molecular marker including a range of CAG repeats should be used in each analysis
Huntington normal vs mutant
Normal: 11-34 CAG repeats
Mutant 42 -> CAG repeats
Triplet repeat mutation diseases
- Fragile X
- Freidrich’s ataxia
- Muscular dystrophy
- Myotonic dystrophy
- Spinobulbar muscular dystrophy
- Spinocerebellar ataxia
- Huntington disease
Detection Hb S steps
1) DNA amplification
2) Digestion with restriction endonuclease
3) Electrophoresis
4) Staining with ethidium-bromide
Indentification of a disease causing gene sequence
1) Linkage
2) Gene localization
3) Gene identification
4) Gene structure and sequence analysis
5) Gene product
* This gives better predictive analysis, better diagnosis and better treatment
Identification sequence of CFTR
1) Linkage:
- Salt transport involvement
- Find a genetic disease feature with known location: DOCRI-917
2) Gene localization
- Tight linkage with RFLP-markers on long arm of chr 7
3) Gene identification
- 920bp long cDNA found from sweat gland
4) Gene structure and sequence analysis
- Sequence show that gene codes for a protein with a transmembrane region
- Expressed in every affected tissue!
- Samples from patients showed triplet deletion (phenylalanine)
5) Gene product
- Chloride ion channel - gene is CFTR
CFTR mutations
- More than 1000 mutations of CFTR gene are known
- Most frequent mutation: del-F508
Method for identification of delF508
Most frequent mutation in CF
1) PCR
2) Allele specific PCR
3) Capillary electrophoresis: Separation of PCR products
NGS
Next generation sequencing assay
- Detection of rare mutations
- MiSeqDx for CF was first FDA-cleared in vitro diagnostic NGS assay
Detection of DNA polymorphism (SNP, VNTR)
- Blot-based indentification: by specific probes (!) (RFLP)
- PCR-based identification: by specific primers (!) - forward and reverse primers
