Lecture 2

Question 1

Before 1953 what did we not know and what did we know?

Answer 1

-We knew that DNA had genetic info did not know structure
- Knew structure thanks to Rosalind Franklin

Question 2

Forward genetics, and what 2 ways can we do it

Answer 2

Starts w/ a phenotype and try to find the genetic basis for observed phenotype Natural→ Mendel’s pea plants
Induced → Flies getting mutated/damaged DNA

Question 3

Mapping

Answer 3

finding location and the number of genes there are

Question 4

Genetic Linkage

Answer 4

traits that show up together are on the SAME chromosome

Question 5

True or false: Recombination of traits on chromosomes are more likely happen the closer they are from one another

Answer 5

False
Traits that RECOMBINE are FARTHER away from one another

Question 6

Sequencing

Answer 6

determining the sequence (order) of bases in genes

Question 7

What is Sanger sequencing (1977) based on?

Answer 7

sequencing based on DNA replication (uses template and primer strand)

Question 8

Sanger Sequencing steps

Answer 8

1. Start with template strand (3’ to 5’) and bind a small complementary primer (5’ to 3’) to it
2. Add polymerase and dNTPs
3. Add ddNTPs
4. Polymerization occurs in 4
5. Tubes get loaded onto gel

Question 9

How were base order determined in Sanger seq?

Answer 9

Used radioactive labled ddNTPs (dideoxynucleotides)
have NO hydroxyl group on 3’ → acts as a chain terminator since nucleotides cannot add to 3’
ddATP →stops after it adds an A
ddCTP→stops after it adds an C
ddGTP→stops after it adds an G
ddTTP→stops after it adds an T

Question 10

How to read Sanger gel?

Answer 10

5’ at bottom of gel, 3’ at top
Read gel from bottom to top (shortest to longest) = 5’ to 3’ sequence → newly synthesized strand
Shortest strand = first base added
For template strand can just do complementary and then write it in 5’ to 3’ direction or read gel top to bottom while at the same time doing complementary of base

Question 11

What was the 1st advancement to Sanger seq?

Answer 11

each base got its own color
A = GREEN
C = BLUE
G = YELLOW
T = RED

Question 12

What was the 2nd advancement to Sanger seq?

Answer 12

instead of using gel, a smaller capillary tube was used (capillary electrophoresis)
Laser would hit base and show a digital output as a peak→computer software says what the base is

Question 13

Limitations of Sanger seq?

Answer 13

-Can only seq 1000 bp at a time –>Signal gets bad with longer DNA strands

Question 14

What limits the length of capillary sequencing?

Answer 14

1. Ratio of ddNTPs : dNTPs
   If not enough ddNTPs then it will not show the positions of every base in sequence (can skip positions) and if too many ddNTPS it will keep stopping the sequence won’t make it too far (will not sequence everything)
2. How well you can separate the DNA lengths
   Signals begin to overlap
   Software guesses what the base is
   If it can’t guess what it is it fills it in with an N

Question 15

What was the human genome sequenced with?

Answer 15

Sanger Sequencing

Question 16

Primer walking strategy

Answer 16

-Top-down strategy (start at 1 end to next)
1. Sequencing must start w/ primer and extend
2. Sequence the primer + extension
3. Build another primer + extension off previous and seq
4. Repeat

Question 17

What is a problem with primer walking?

Answer 17

can’t multiplex, must wait for the previous seq in order to do next

Question 18

True or false: Human genome was sequenced using primer walking method

Answer 18

False
-would take too long (8,000+ years)