Unidentified disease gene

Question 1

Approaches used to identify unidentified disease gene

Answer 1

1. Biochemical detect approach
2. Position- independent candidate gene approach
3. Linkage Analysis approach
4. Positional Candidate approach

Question 2

How are candidate genes selected?

Answer 2

Selected based on their functional relevance to the disease phenotype without knowledge of genomic location

Question 3

What is the positional candidate approach?

Answer 3

Positional information from linkage analysis and functional insights into gene expression patterns

Question 4

Sanger sequencing

Answer 4

Uses dideoxynucleotide: causes termination of the reaction at specific points

Question 5

Next-Generation Sequencing NGS

Answer 5

Relies on sequencing DNA through synthesis, where nucleotides are incorporated into the growing DNA strand and can be monitored in real-time as they are added

Question 6

Pyrosequencing

Answer 6

Method that tracks the sequencing process by monitoring the polymerisation process

Question 7

Method used by pyrosequencing

Answer 7

Release of Pyrophosphate when a nucleotide is added to a DNA strand

Question 8

Illumina Sequencing process

Answer 8

1. Fragment the DNA and bind adaptors that have chips
2. Amplification of the copies of each fragment
3. Sequencing

Question 9

Overview of Illumina Sequencing

Answer 9

Add the first rounds of nucleotides, remove the unincorporated, detect the signal, deprotect the 3’ end and so the second round of sequencing

Question 10

Pacific Biosciences ZMW

Answer 10

allows sequences up to kilobases in a single reaction unlike the other 2 methods

Question 11

Pacific Biosciences ZMW technique

Answer 11

Involves a SMRT chip which contains thousands of perforations in which DNA polymerase performs DNA sequencing with a single DNA molecule template

Question 12

Basis of Pacific Biosciences ZMW

Answer 12

the nucleotides are labelled using fluorophore on the phosphate group ; not the base. There is a little chamber of excitation and emission so that emission signal of the difluorophore just before it is incorporated in the DNA can be detected

Question 13

Capture-based sequencing

Answer 13

Only sequence a subset of the fragments that compose the genome; these subsets could be whole exome sequencing or panels for targeted sequencing

Question 14

Functional analysis

Answer 14

Performing exon sequencing only for the genes involved in the function of a particular type of protein

Question 14

Candidate exome capture

Answer 14

combining exome sequencing and linkage analysis

Question 15

Strong evidence of pathogenicity

Answer 15

Predictive null variant in a gene where loss of function is a known mechanism of disease
De novo mutation

Question 16

What can exome sequencing do?

Answer 16

Repurpose or redirect the clinical diagnosis

Question 17

How was the Bardet Biedl Syndrome investigated?

Answer 17

Genes present in organisms that have a primary cilium or organelles similar to the primary cilia were used: comparison of genes shared between organisms that had the primary ciliium and organism that does not have this organelle: brought 688 predicted proteins

Question 18

Method of positional cloning approach

Answer 18

1. Dissect what is going on in a population
2. Identify biochemical pathways
3. identify what is going on molecularly
4. Build a list of possible candidate genes, whose function is involved in these activities that are affected

Question 19

Validations to support that the 688 proteins were involved with primary cilia

Answer 19

1. Should contain many of the known genes that have a role in the primary cilium
2. The domain characteristics of the proteome should be consistent with the domains of the known flagellas and basal body proteins
3. A substantial fraction of the genes should be upregulated after deflagellation
4. Ablation of the genes should cause flagellar/basal body phenotypes
5. The proteome should contain genes involved in disorders of human ciliation