Lecture 25

Question 1

How are normal cells different than cancer cells?

Answer 1

• Cancer cells have a large, variably shaped nuclei
• Cancer cells divide frequently in a disorganized arrangement
• Cancer cells vary in size and shape
• Cancer cells lose normal features

Question 2

What are the three stages of cancer?

Answer 2

• Elimination by the immune system
• Equilibrium is kept by the immune system, tumor is dormant
• Escape from the immune system, progressive disease

Question 3

How did the original sequencing occur?

Answer 3

Sanger sequencing

• A single stranded DNA is labelled with a primer.
• DNA polymerase adds the complementary base from the excess dNTPs
• Each binding of dNTP stops the reaction -various sized fragment
• the fragments are run on a gel based on sizes
• gel is red from 5’ to 3’ end to determine the sequence

Question 4

What happens if there is no 3’-OH?

Answer 4

• 3’-OH required for chain elongation
• if there is no 3’-OH then the chain terminates
• bases are added to the 3’ end
• could be removed due to mutation

Question 5

Why is Sanger sequencing helpful?

Answer 5

• Allows you to see mutations which are visualized on the gel
  ie. a heterozygous point mutation where there is a wild-type copy of the gene and a mutant copy of the gene in the same location

Question 6

How was Sanger sequencing updated?

Answer 6

• The dNTPs were labelled with fluorescence
• The fluorescence is then detected by a computer
• Peaks are generated which correspond to a base
• Can sequence 1000 bases with accuracy

Question 7

How has sequencing the human genome changed in the past 10 years?

Answer 7

• In 2006, the cost to sequence a single human genome was $10 million
• In 2015, to sequence more than 10 million human genomes cost less than $100

Question 8

How is the genomic DNA library prepared for Next Generation Sequencing?

Answer 8

• The DNA fragment has its ends sheared, then polished to make them blunt ended
• The fragment is ligated to adapters
• It is then separated on a gel then cut out

Question 9

What does a flow cell look like?

Answer 9

• There are 8 channels

- The surface of the flow cell is coated with a lawn of oligo pairs

Question 10

How does cluster generation begin?

Answer 10

• Begins with hyridization of the fragment to the oligos
• > 100M single molecules hybridize to the lawn of primers
• Bound molecules are then extended by polymerases

Question 11

After the formation of the newly synthesized strand, what occurs during Next Generation?

Answer 11

• The double-stranded molecule is denatured
• The original template is washed away
• The newly synthesized strand is covalently bound to the flow cell surface

Question 12

What is bridge amplification in Next Gen sequencing?

Answer 12

• Single strand flips over to hybridize to adjacent primers to form a bridge
• Hybridized primer is extended by polymerases
• Double-stranded bridge is formed
• “PCR on a solid support” -> generates thousands of copies to provide enough signal during sequencing

Question 13

What occurs in Next Gen once bridge amplification is completed?

Answer 13

• The double stranded bridge is denatured
• Results in two copies of covalently bound single-stranded templates
• these templates undergo the bridge amplification again until multiple bridges are formed
• they are then denatured and the reverse strands cleaved and washed away

Question 14

How does the sequencing cycle in Next Gen occur?

Answer 14

• Determine the first base by adding the four labelled dNTPs

- Excited by a laser and the bases light up in their location

Question 15

How are gaps filled in Next Gen?

Answer 15

Using a reference sequence, the fragment sequences are aligned and the gaps are filled by overlays

Question 16

What is high coverage?

Answer 16

When there is a large amount of the genome sequenced

• multiple fragments that cover various parts of the genome
• more accurate

Question 17

What is low coverage?

Answer 17

When there is a small amount of genome sequenced

- could leave gaps

Question 18

What are the differences between Sanger Sequencing and Next generation Sequencing?

Answer 18

• Sanger uses a primer while NG uses oligos
• Sanger individually interogates each terminated DNA while NG uses a DNA library
• Less info is gathered by Sanger and NG gathers more info

Question 19

What could be missed from Whole-exome sequencing?

Answer 19

You would miss regulatory elements, such as microRNA, start sites, etc, that are within introns

Question 20

What is whole-exome sequencing used for?

Answer 20

• point mutations

- copy number variations

Question 21

What is exome sequencing?

Answer 21

• sequences only protein-encoding genes (exons, 1% of the genome)
• looking at exons because they’re responsible for phenotypes in a cell

Question 22

What are the limitations of exome sequencing?

Answer 22

• only accounts for 1% of the genome (~180K exons)

- misses deletions, variants and gene-rearrangements

Question 23

What are the advantages to exome sequencing?

Answer 23

• cost effective
• quick
• can see point mutations
• can look at copy number variations

Question 24

What is upregulated by at T-cell that has become active?

Answer 24

• 4-1BB

- IFNg

Question 25

Why is personalized medicine important?

Answer 25

- all cancers are genetically different with different mutations

Question 26

How is a person with metastatic cholangiocarcinoma treated with T cells? What are the results?

Answer 26

- A biopsy was taken of the patient's cancer - extracted T cells that recognized the tumor and grew up the numbers - The mutation was found in the ERBB2IP protein by T cells - Results: T cell therapy induced regression at 6 months but post 6 months there was an increase in tumor burden again. This could be due to other mutations

Question 27

What cancer applications are there that have T cells that identify targets?

Answer 27

- Adoptive T cell therapy - Vaccines - Chimeric antigen receptors - Engineered T cells