T1: Genome Analysis Methods

Question 1

What are the main laboratory techniques for investigating the genome?

Answer 1

• PCR
• Sanger sequencing
• Next-generation sequencing
• Cytogenetic testing techniques

Question 2

What is PCR used for?

Answer 2

PCR amplifies the target DNA. It is used to detect the presence of specific sequences i.e. genomic sequences, virus’ and circulating foetal and tumour DNA. It can generate a template for other analysis and sequencing.

Question 3

What is Next-Generation Sequencing?

Answer 3

NGS technologies include illumina sequencing technology. It uses clonal sequencing machines. Clones are generated using PCR onto a flow cell. Each clone is sequenced similar to that is sanger sequencing. The florescent dye is cleaved off. They can have a removable terminator. Once we have incorporated the next base we can attach another dye added. Shine a laser and we slowly build up the sequences. The sequencing reads tend to be quite small. It is used as Sanger sequencing is quite expensive and time consuming.

Question 4

Give common NGS sequencing projects.

Answer 4

Long range PCR products - diagnostics
Targeted capture gene panels - diagnostics
Whole exome sequencing (WES) – research/diagnostics
Whole genome sequencing (WGS) – research/diagnostics

Question 5

What techniques are used to test the whole genome?

Answer 5

• Sanger Sequencing
• Next generation sequencing
• Microarrays
• G-banding (Traditionally we look at chromosomes at halt them in metaphase and karyotype. Then identify them using g- banding. This is very time consuming. It has variable resolution. Here we can see changes in chromosome number).

Question 6

What techniques are used for targeted testing?

Answer 6

• FISH
• MLPA
• QF-PCR

Question 7

What is Array CGH?

Answer 7

Array CGH is a technique which screens the whole genome to detect copy number changes (unbalanced gains/duplications and losses/deletions of genetic material) which may be contributing to a child’s phenotype. Array CGH will not detect balanced chromosome rearrangements, such as balanced translocations or inversions.

Sample DNA (with green tags) and the reference RNA (with red tags) are hybridised to an array with lots of probes on it that span the genome. Depending on the intensity of the brightness, if there is an equal amount of DNA it will show yellow. If there is more of the sample DNA, it will show green and red if there is more reference DNA.

Question 8

What is the difference between cytogenetic techniques and molecular analysis techniques?

Answer 8

Cytogenetics involves the examination of chromosomes to identify structural abnormalities. Fluorescent in situ hybridization (FISH) is a process that vividly paints chromosomes or portions of chromosomes with fluorescent molecules to identify chromosomal abnormalities (e.g., insertions, deletions, translocations, and amplifications).

Direct DNA analysis is applicable when the gene sequence of interest is known. For small DNA mutations, direct DNA testing is typically the most effective method, particularly if the function of the protein is unknown and a biochemical test cannot be developed. A DNA test can be performed on any tissue sample and requires very small amounts of sample. Several different molecular technologies, including direct sequencing, polymerase chain reaction-based assays (PCR), and hybridization, can be used to perform testing.