Rare disease genetic testing Flashcards
What is Sanger sequencing? (4)
- First type of sequencing
- Requires a PCR primer set for every exon
- Time consuming and resource heavy because you sequence one gene at a time
- Used now to sequence single exons for familial testing
What are the 2 types of microarrays?
- Oligonucleotide microarrays
- Single nucleotide polymorphism arrays
What are oligonucleotide microarrays? (3)
- Detects gain or loss of patient DNA vs control (competitive hybridisation)
- 0 = normal
- > 0.5 = gain of patient DNA
- <-0.5 = loss of patient DNA
What are single nucleotide polymorphism arrays? (3)
- Analysing normal polymorphisms
- Probe for possible polymorphisms, probes bind to the patient DNA
- Amount of binding tells you homozygous AA/BB or heterozygous A/B
What is genomic sequencing? (2)
- (Next generation sequencing)
- DNA is sequenced many times rather than just once in Sanger, more accurate
How much of the genome is protein-coding?
1%
What are the types of genomic test? (4)
- Single gene sequencing (Sanger)
- Targeted gene panels (a set of genes linked to a particular condition)
- Exomes (NGS for protein-coding only)
- Genomes (NGS of the whole genome)
What are the benefits of NGS? (4)
- Allows analysis of thousands of genes in one assay
- Whole genome sequencing enabled
- Can run multiple patients in one assay
- Many more diseases diagnosed via one test
What are the types of DNA variants? (8)
- STOP and START variants
- Missense
- Nonsense
- Synonymous (doesn’t change the amino acids)
- Splice site
- Duplications/deletions (frameshift)
- May be a single nucleotide change
- May be multiple nucleotide changes
What is the start sequence?
ATG coding for methionine
What is the most common variant?
Missense - changes the amino acid that is being coded for
Which nonsense variants may escape NMD? (2)
- DNA variant is present in the last exon
- DNA variant is located in the last 50 nucleotides of the penultimate exon
What is a copy number variant (CNV)?
Large deletions/duplications that encompass more than one gene
What is the acceptor splice site? (2)
- The 5’ end of the intron
- Always an AG dinucleotide
What is the donor splice site? (2)
- The 5’ end of the intron
- Always a GU dinucleotide
How does splicing occur? (3)
- Small nuclear RNA (snRNA) molecules bind to specific proteins forming a small nuclear ribonucleoprotein complex (snRNP)
- snRNPs combine to form the spliceosome
- Spliceosome identifies the acceptor and donor splice sites and removes the intron
What are splice variants? (4)
- Variants that occur in introns
- If the variant occurs in the donor splice site the intron won’t be removed which changes the protein sequence and can cause alterations to the reading frame
- Variant in the acceptor splice site means a whole exon is removed which may be vital to the protein function
- Splice changes are often disease causing
What are the symbols for each type of DNA sequence? (5)
- c. = coding DNA (includes introns)
- g. = genomic sequence
- m. = mitochondrial DNA
- r. = RNA
- p. = protein (amino acid) sequence
What can variants be in terms of alleles? (3)
- Heterozygous (one allele has the variant)
- Homozygous (both alleles have the variant)
- Hemizygous (if X-linked and a male carrier)
