Genetics 7 Flashcards
(52 cards)
Describe the characteristics of monogenic disease
Monogenic disease: one mutation
Monogenic disease: one gene, many mutations
Describe the characteristics of polygenic disease
Multigenic disease: many genes, many mutations
What are the principles of a DNA test
DNA isolation
PCR (amplification of selected DNA region) + digestion, if needed
Visualisation of result on an agarose gel
Describe the use of PCR in the diagnosis of cystic fibrosis
PCR with primers that span region of CFTR gene for 3bp deletion that results in ΔF508
The mutant product is 3 nucleotides shorter than the normal product.
How can we use PCR to detect single nucleotide changes
PCR- amplify products
Use specific restriction endonucleases that cleave the DNA at specific sites ( the site of a mutation)
New fragment in homozygote phenotype of disease
new fragment + normal fragment in heterozygote phenotype.
This is known as Restriction Fragment Length Polymorphism
Describe PCR multiplexing
Allele-specific PCR
Mutation specific primers: Primers bind to mutant DNA- oligonucleotides and DNA ligase are used to join the two primers- creating a PCR amplifiable molecules
What do we do if we don’t know the mutation
We sequence all of the exons in the gene- Snager sequencing to look for point mutations in the gene.
How does sanger sequencing work
Single-stranded PCR product
Add primer, DNA polymerase and 4 didieoxy nucloetides
When they bind to the template strand by complementary base pairing, they terminate the growing chain as they have no 3’-OH group.
The bases are tagged with a specific colour
The fragments each ending in a different base are separated by capillary electrophoresis- to get size order
Chromatogram reads the colour of each fragment
Sequence is determined
Why is it important to get the right amount of chain terminators.
More terminator A than A:
Strand would stop growing at a specific base length, and so would accumulate
Bands of certain base lengths would not be synthesised.
What are the pros and cons of Sanger Sequencing
Pros ; Low error rate, long read length
Cons: Expensive, need a lot of DNA to start, Can only do 1 sequence at a time, can only do one forwards and one reverse reads.
What are the pros and cons of Next Generation sequencing
Pros- fast turnaround time, massively parallel (millions of fragments can be sequenced in a single run), cheaper.
Cons: Short read length, fewer reads of each bases are combined, so less accurate overall.
Not targeted- unlike Sanger sequencing
Describe the process of Next Generation Sequencing
Genomic DNA from the patient is fragmented, denatured and exposed to a cocktail of probes that represent desired sequences to check. The hybridizing fragments are isolated and used for sequencing.
The fragments are then aligned to a reference genome
Sequences line up, same base as that of reference, may line up but be different base- heterozygote
Describe the importance of long read lengths
In order to make sense of the myriad of short reads generated, they must be aligned to the reference genome, the shorter the individual reads, the harder it is to align them unambiguously, especially in repetitive regions of the genome.
Describe the importance of read depths
To distinguish true variants from random sequencing errors. Hence, we need a large number of independent reads across any genomic position. If there are only two good reads, one G and one A, impossible to know which one is a true variation or sequence error. 14As and 1G- obvious. 9As and 6Gs- heterozygous.
Describe the different library preparations
Genomic – DNA Whole genome Targetted Exome sequencing (1% genome) Gene panels Epigenome (DNA methylation sites) Transcriptomic – (polyA) mRNA Short regulatory RNA Protein bound regions Transcription factors Protein-mRNA interactions
Describe the difference between whole genome and targeted capture
Whole genome- sequence whole genome to get target gene output from one flow cell
Targeted- enrich targeted DNA- output from one lane flow
This is cheaper.
What are gene panels used for
Sequence selected sets of genes:
Multigenic diseases, e g Primary Ciliary Dyskinesia (PCD)
Mutations identified in 37 genes -> commercial gene panel
Detects 80% of mutations
300 candidate genes with an identified role in ciliogenesis -> gene panel for identification of novel mutations
Population specific gene panels
What is the purpose of epigenome analysis
To see which parts of the genome are active
Describe how we analyse the epigenome
Treat the DNA with bisulfite under carefully controlled conditions.
Bisulfite converts unmethylated cytosines into uracil by deamination.
Hybridise
Amplify by PCR
Sequence by NGS or Sanger Sequencing
Every unmethylated C will appear as a T, allowing us to analyse methylation patterns.
What is the purpose of analysing the transcriptome
Which genes are active in cell/tissue?
Gene expression differences in healthy/affected tissue
-> Biological pathways involved in disease
Can RNA be cloned, amplified or sequenced
No
Describe how we analyse the transcriptome
Collect sample of mRNA
Convert into cDNA using reverse transcriptase
Amplify by RT-PCR- using specific primers.
What is the difficulty of using RNA
RNA is harder to obtain and handle than DNA.
Mutant genes often produce no detectable mRNA.
RNA is unstable, requiring stringent conditions in the laboratories- need special reagents to stabilise RNA.
Only expressed in specific tissues.
What is the purpose of short regulatory RNA, miRNA sequencing
Which genes are silenced by short interfering RNA?
Identification of regulatory miRNA for gene silencing e.g oncogenes
