Human Genome 2

Question 1

What is dideoxy sequencing (Sanger sequencing)?

Answer 1

• lacks 3’ OH group
• highest accuracy, but does one sequence at a time

Question 2

What are the steps of manual dideoxy sequencing (1st gen)?

Answer 2

1. single stranded DNA fragment
2. attach with short DNA primer
3. divided into 4 test tubes -> one for each each base
4. add DNA nucleotides to test tubes with small amount of dideoxy nucleotides to stop W/C pairing
5. multiple fragments with different lengths
6. gel electro. to separate strand by size

Question 3

What is 2nd generation sequencing?

Answer 3

• takes longer than 1st gen
• allow multiple genomes to be sequence in parallel
• good for finding mutations

Question 4

What are the steps of illumina sequencing?

Answer 4

1. add fluorescence (no WC bp change)
2. add primers and nuncleotides with blocked 3’ OH
3. take pic of nucleotide that sticks
4. remove blocked 3’OH
5. add color back and repeat

Question 5

What are the steps of massive parallel sequencing (next gen)?

Answer 5

1. fragment DNA (150 bp)
2. separate strandns
3. add primers to both ends
4. place strands on chip with extra primers
5. segments form a bridge
6. DNA polymerase creates double strand DNA
7. denature double strand into single
8. sequence DNA one at a time
9. overlap to get sequence

Question 6

In massive parallel sequencing, how are primers bonded to the chip?

Answer 6

covalently

Question 7

What is whole genome sequencing (shotgun)?

Answer 7

fragment whole genome and stitch overlaps to get whole genome

Question 8

Exomes make up what percent of the genome?

Answer 8

2%

Question 9

What are the steps for whole genome sequencing?

Answer 9

1. link DNA fragment to bacterial DNA
2. allow bacteria to grow and replicate DNA
3. isolate DNA
4. can do massive parallel to determine full sequence

Question 10

What are contigs?

Answer 10

• large section of chromosome
• when entire sequence in complete with one contig per chromosome

Question 11

What is a BAC?

Answer 11

• modified plasmid vector that allows large fragments to be stably propagated
• up to 500 nt for overlapping
• cultured in liquid

Question 12

What was the main point in the evolution of cancer? (article)

Answer 12

• somatic evolution can lead to cancer by altering things that increase proliferation
• caused by alteration in DNA sequences
• use next gen & massive parallel