Part E: Lecture 36

Question 1

Timeline for DNA sequencing

Answer 1

Manual Sanger (1970s)
Automated Sanger (1980s)
Next Gen (2008)

Question 2

Automated Sanger machine

Answer 2

ABI 3730

Question 3

Next Gen machine

Answer 3

Illumina MiSeq
Illumina NextSeq 500

Question 4

Automated Sanger Results

Answer 4

fluorescent coloured peaks for nucleotides (chromatogram)

Question 5

Next Gen Results

Answer 5

list of letters corresponding to nucleotides (no chromatogram)

Question 6

M. Sanger _____ by A. Sanger and now it is used with _____

Answer 6

replaced
Next Gen

Question 7

dideoxynucleotides (ddNTPs) description

Answer 7

no 3’ OH

Question 8

ddNTPs function

Answer 8

stop DNA replication

Question 9

Cy3 emits ___ light

Answer 9

greenish-yellow

Question 10

Cy5 emits ___ light

Answer 10

red

Question 11

ddNTPs is connected to ____ to indicates ____

Answer 11

fluorescent dyes
nucleotide at specific position

Question 12

DNA sequencing reaction mixture

Answer 12

Template DNA
one oligo primer
Taq DNA Pol
lots of normal dNTPs
some labeled ddNTPs

Question 13

DNA sequencing steps

Answer 13

1) rxn mixture
2) in vitro DNA synthesis
3) Capillary electrophoresis

Question 14

Machine for DNA sequencing

Answer 14

standard PCR machine

Question 15

number of cycles of denature/anneal/extend for DNA sequencing

Answer 15

25 cycles

Question 16

capillary electrophoresis

Answer 16

(-) electrode and (+) electrode connected via capillary tube which separates dna fragments according to size (smallest is first), laser fluoresces DNA and detector detects colour

Question 17

DNA sequencing results

Answer 17

Chromatogram

Question 18

useful data (range) for DNA sequencing

Answer 18

700 nt

Question 19

PCR vs. A. Sanger: product shape

Answer 19

PCR: dsDNA of equal length
A. Sanger: ssDNAs of unequal length

Question 20

PCR vs. A. Sanger: product number

Answer 20

PCR: 1 billion products per template
A. Sanger: 25 products per template

Question 21

PCR vs. A. Sanger: product max size

Answer 21

PCR: 5kb
A. Sanger: 700 nt

Question 22

A. Sanger sequencing is used for _____

Answer 22

sequencing plasmids and genes

Question 23

To make primers, we need to know ______

Answer 23

the sequence

Question 24

sequencing a plasmid steps

Answer 24

1)isolate plasmid DNA
2)sequence with primer

Question 25

sequencing a gene steps

Answer 25

1)isolate genomic DNA
2)PCR to amplify gene
3)sequencing with primer

Question 26

PCR vs. A. Sanger: # primers

Answer 26

PCR: 2 primers
A. Sanger: 1 primer

Question 27

DNA sequencing chromatogram: heterozygous

Answer 27

lower double peak