19.2

Question 1

principles of DNA sequencing

Answer 1

the highest resolution map is the base pair sequence
-various methods of sequencing rely on:
*PCR
*electrophoresis
*chain-terminating nucleotides
-Manual sequencing discovered by Fred Sanger

Question 2

Dideoxy (automated) sequencing

Answer 2

1. start with strand of unknown sequence
2. amplify using PCR
3. incorporate dideoxynucleotides
   *labeled with fluorescent marker for A,T,C,G
4. fragments separated by electrophoresis
   *smallest fragments (5’ end) migrate fastest
5. utilize laser and photodetector to identify base

Question 3

next generation sequencing

Answer 3

is faster, cheaper, and has ability to seuence larger fragments of DNA
-utilizes DNA fragments attached to solid surface
-multiple copies of fragments ensure accuracy
-uses “reversible” chain terminating nucleotides

Question 4

sequences genome fragments are assembled into complete sequences

Answer 4

most genomes are too large to be sequenced in a single step. must be broken into manageable fragments
1. clone-contig method
2. shotgun mehtod

Question 5

clone-contig method

Answer 5

breaks genome into large “clone” fragments

Question 6

shotgun method

Answer 6

whole genome is fragmented into manageable pieces
rely on computers to find overlap and reassemble

Question 7

human genome project

Answer 7

draft sequence established in 2020
more than 20 years of work
found 20,000 genes—expected 100,000
organismal complexity is not merely a function of genome size or gene number