Lecture 1: Sanger Sequencing

Question 1

What are the four different approaches to DNA sequencing?

Answer 1

Chemical degradation of DNA

Sequencing by synthesis (SBS)

Ligation-based

Nanopore

Question 2

Talk about the chemical degradation apprach to sequencing

Answer 2

The first method of DNA sequencing
Not carried out anymore
Uses too many dangerous chemicals

Question 3

Talk about the sequencing by synthesis (SBS) method of DNA sequencing

Answer 3

The most common approach to sequencing
Developed by Sanger, also known as Sanger sequencing
Works by primer extension through the use of DNA polymerase and nucleotides
DNA must be denatured into single strands first, primer must be annealed and then oligonucleotides added

Question 4

Talk about ligation-based methods of DNA sequencing

Answer 4

Sequencing using short probes that hybridise to the template

Question 5

Talk about using nanopores as a sequencing method

Answer 5

The newest way of sequencing DNA
Inferring sequence by change in electrical current as ssDNA is pulled through a nanopore

Question 6

Briefly describe the structure of DNA nucelotide

Answer 6

Consists of a 5 carbon sugar with a nitrogenous base attached at the 1’ C and a phosphate group attached to the 5’ carbon

A hydroxyl group is present on the 3’ carbon

Question 7

What are the different nitrogenous bases in DNA vs RNA

Answer 7

Adenine, Guanine, Cytosine

+ Thymine in DNA
+ Uracil in RNA

Question 8

How does uracil differ from thymine?

Answer 8

Uracil lacks a methyl group

Question 9

Talk about the hydrogen bonds between G and C and A and T and why they are important to consider

Answer 9

Adenine and Thymine form two hydrogen bonds to hold together
Guanine and cytosine form three hydrogen bonds to hold together

This is important to consider as you may have to heat a GC bond rich segment of DNA to higher temperatures to get it to denature

Question 10

How does ribose differ from deoxyribose

Answer 10

The 2’C in ribose has a hydroxyl group attached

The 2’ C in deoxyribose lacks an oxygen (hence deoxy) and only has a H attached

Question 11

Describe the directional structure of DNA and why this is important

Answer 11

DNA is an anti-parallel structure i.e. its two strands are in opposite directions, 1 strand in the 5’ to 3’ direction and the other in the 3’ to 5’ direction

DNA polymerase can only work in the 5’ to 3’ direction ie. a strand can only be built in the 5’ to 3’ drection

Question 12

What bonds are formed to join oligonucleotides to form a strand of DNA and how are they formed

Answer 12

Phosphodiester bonds

These bonds are formed between the phosphate of one nucleotide and the 3’ hydroxyl group of another

There is the loss of a hydrogen ion with the formation of a phosphodiester bond (this H loss can be detected and used in some other forms of sequencing)

Question 13

How does modern day Sanger sequencing differ from when it was first used

Answer 13

When Sanger was first used to sequence the human genome plasmid clones were used to create multiple clones of a sequence, now we use PCR to amplify up multiple copies of the DNA before sequencing

Question 14

How does modern day Sanger sequencing differ from when it was first used

Answer 14

When Sanger was first used to sequence the human genome plasmid clones were used to create multiple clones of a sequence, now we use PCR to amplify up multiple copies of the DNA before sequencing

Question 15

What strand acts as the template for sanger?

Answer 15

Reverse strand acts as the template for sanger
Template is 3’ to 5’ direction so that nucleotides can be added on in the 5’ to 3’ complementary to that

Question 16

What are chain terminators

Answer 16

Dideoxy nucleotides i.e. DNA nucleotides which lack their 3’ OH and therefore cannot be further extended (cannot form phosphodiester bonds)

They are used to terminate a sequence chain

Question 17

How do chain terminators work?

Answer 17

Removal or modification of the 3’ hydroxyl group needed in the formation of phosphodiester bonds prevents DNA polymerase from incorporating any further nucleotides, thus the sequence ends

Dideoxy nucleotides are the chain terminators used in sanger sequencing, these lack an oxygen on their 3’ hydroxyl group (i.e. they have only a 2H on their 3’) which terminates the reaction

Question 18

How was Sanger sequencing originally carried out using dideoxynucleotides

Answer 18

Sanger set up 4 individual reactions each containing a mixture of DNA nucleotides as well as a small concentration of dideoxynucleotides (either ddTTP, ddATP, ddGTP or ddCTP)

The presence of low concentrations of a ddTP meant there was a low chance of the reaction randomly stopping which produces DNA sequences of different lenghts

A polyacrylamide gel was then used to separate out the DNA sequences according to their weight

From a single fragment of DNA we know only the identity of the last nucleotide in the sequencing but when all are combined in a gel we can read the order of the nucleotides to determine the sequence

Question 19

How did Sanger visualise his sequence

Answer 19

Sanger used radioactive signals -> he used 32P as a radioactive label

Gel was exposed to an x-ray film to make an auto-radiogram

Question 20

Why use polyacrylamide gel and not just agarose gel?

Answer 20

Polyacrylamide gel allows one to differentiate DNA fragments down to the individual base pais e.g. allows one to tell between a 99 base pair chain and a 100 base pair chain

Question 21

Why use polyacrylamide gel and not just agarose gel?

Answer 21

Polyacrylamide gel allows one to differentiate DNA fragments down to the individual base pais e.g. allows one to tell between a 99 base pair chain and a 100 base pair chain

Question 22

What kind of primer must be used in Sanger sequencing?

Answer 22

A primer with a nucleotide sequence complementary to the 3’ end of the region to be copied (reference)

Question 23

What do you need to set up a Sanger sequencing reaction?

Answer 23

Template DNA
Nucleotides
Dideoxyucleotides
Buffer
DNA polymerase
Forward or reverse primers

Question 24

What do you need to set up a Sanger sequencing reaction?

Answer 24

Template DNA
Nucleotides
Dideoxyucleotides
Buffer
DNA polymerase
Forward or reverse primers

Question 25

What happens when a dideoxynucleotide is added?

Answer 25

Replication of the strand will stop i.e. terminate

Question 26

What is the difference between a dideoxynucleotide and a deoxynucleotde?

Answer 26

A dideoxynucleotide is missing a 3’ hydroxyl group on its ribose

Question 27

What were some areas that needed improvement in Sanger sequencing?

Answer 27

Had to be ran on a very large gel -> preferred if it could all be done on one lane to save

Would be better if signals could be read at the same point in the gel -> youre unable to read the sequence near the top as the bands get closer and closer together

Fluorescent labels would be safer and easier to read then radioactive labels -> can also use different fluorescent labels of different colours which can be read by a laser

Question 28

What was the imporoved Sanger sequencing called?

Answer 28

Fluorescent Sanger Sequencing “Dye terminators”

Question 29

How does Fluorescent Sanger Sequencing differ from normal Sanger?

Answer 29

Each of the 4 ddNTPs are labelled with a different fluorescent dye instead of a radioactive label

Fluorophores added to ddNTPs -> this made the ddNTPs much bigger though so a modified DNA polymerase had to be used

Question 30

What imporovement was made to fluorescent sanger sequencing?

Answer 30

Caltec automated the process in the mid1980s by addition of a gel in a capillary and a laser

The process is ran as normal except instead of using a large gel a mini gel contained inside of a capillary is used

A laser is then used to read the fluorescent signals

Question 31

How is fluorescent sanger sequencing interpreted?

Answer 31

You get four signals of four different colours, one for each type of ddNTP that has been incorporated

The laser reads the position and colour of the signal, this is interpretted by a base caller system to provide us with a readable trace (sequence)

Question 32

What is Sanger Base Calling?

Answer 32

A software which provides an estimation of the quality of a sanger sequence -> a software which tells us the possibility that a base has been interpreted incorrecly -> possibility of error

Question 33

Talk about the quality of a sanger sequence
(2)

Answer 33

Poor quality in the first 15-40 bases of a sequence due to primer binding

Deteriorating quality of sequence traces after the 700-900 bases due to polymerase gradually loosing steam i.e. loosing its ability to add on nucleotides

Question 34

What base calling software do we use?

Answer 34

Phred

Question 35

What base calling software do we use?

Answer 35

Phred

Question 36

Explain in your own words what a base calling software like Phred does?

Answer 36

Software which interprets the fluorescence and names the base it corresponds with

Software which calculates how accurate a base pair read is i.e. how correct a fluorescence has been labelled

Question 37

What is the name of the score we use to determine how much we can trust a sanger sequence read?

Answer 37

Q scores

Question 38

How do you interpret a Q-score?

Answer 38

You want a Q score as close to 99 as possible

Dont trust any Q score less than 20

Q20 = a 1 in 100 chance the base is incorrect
Q99 = 1 in 10^-10 chance the base is incorrect

Question 39

What is CHOPS syndrome?

Answer 39

Cognitive impairment
Heart defects
Obesity
Pulmonary Involvement
Short Stature
Skeletal dysplasia

Question 40

Mutations of what gene are responsible for CHOPS?

Answer 40

AFF4 mutations

Question 41

Explain how you would read fluorescent sanger results when looking for a mutation e.g. AFF4 mutations in chops

Answer 41

Three known dominant mutations of AFF4 cause chops

Sequence these regions

Look for two peaks at the one signal -> this indicates one normal copy and one mutated copy

*if the condition was recessive you would need two copies of the mutated gene or a combination of mutated genes etc etc

Question 42

How has sanger sequencing improved?

Answer 42

Moved from polyacrylamide gels and radioactive labels to capillary gels and fluorescent labels

Used to be read by hand but are now automatically read by a laser

Low throughput on old methods, would have taken years to squence whole genome, now you can sequence 1-2 million bp/day using sanger