Lecture 21

Question 1

When and by who was manual Sanger DNA sequencing made?

Answer 1

1977, Frederick Sanger

Question 2

When was automated Sanger DNA sequencing made? What does it use?

Answer 2

1986, ABI 3730 (makes peaks)

Question 3

What are the materials required for Sanger sequencing? What do they do?

Answer 3

dideoxynucleotides (ddNTPs): terminate DNA replication

fluorescent dyes, labels each ddNTP with a unique dye colour

Question 4

Why does ddNTPs terminate DNA replication?

Answer 4

lacks a 3’ OH group

Question 5

What colour does the fluorescent dye Cy3 emit?

Answer 5

green-yellow

Question 6

What colour does the fluorescent dye Cy5 emit?

Answer 6

red light

Question 7

What are the 4 ddNTP dye combinations?

Answer 7

ddATP: green
ddGTP:yellow
ddCTP: blue
ddTTP: red

Question 8

What is the Sanger procedure?

Answer 8

1) either sequence a plasmid or PCR product

2) reaction mixture, with template DNA, one oligo, Taq DNA polym, lots of normal dNTPs, small amount of fluorescent ddNTPs

3) cycling: run ~25 cycles in thermocycler, produces mixture of DNA fragments that end at every possible position

4) capillary electrophoresis, uses ABI 3730, separates fragments by size and reads fluorescence

5) data interpretation (in chromatogram

Question 9

What are the differences between PCR and auto Sanger?

(What do they process, what type of dNTP, do they terminate, how to visualize,what do they see, how many primers)

Answer 9

PCR: amplify specific DNA fragment, uses ONLY NORMAL dNTPs, never terminates early, visualized by agarose gel electrophoresis, sees presence/absence/size of product, uses standard thermal cycler, requires 2 primers

Auto Sanger: determines NT sequence of DNA, uses dNTPs + small amount of ddNTPs (terminators), visualized by chromatogram, sees base by base sequence, uses capillary electrophoresis sequencer, uses ONE PRIMER, reads sequence in one direction

Question 10

How many primers does PCR have?

Answer 10

2

Question 11

How many primers does auto Sanger have?

Answer 11

1

Question 12

What is the procedure in sequencing plasmids?

Answer 12

use primers flanking the insert

Question 13

What is the procedure in sequencing genes?

Answer 13

use gene-specific primers (left or right), such as seuqnecing around a start codon

Question 14

Which peak is higher, homo or heterozygous?

Answer 14

Homozygous

Question 15

What is next-gen sequencing?

Answer 15

sequence large amounts of DNA fast and cheap

Illumina MisSeq and Illumina NextSeq 500

Question 16

What is the process of NGS?

Answer 16

Obtain DNA (plasmid or PCR product)

send to plasmidsaurus

all samples separated by sample indexing