Lecture 3: Studying Human DNA: DNA sequencing (not finished)

Question 1

what san example of a sequence polymorphisms ?

Answer 1

single nucleotide polymorphisms (SNPs)

Question 2

what does DNA sequencing determine?

Answer 2

DNA sequencing determine the precise order of nucleotides.

Question 3

is there more than one method to sequence DNA?

Answer 3

yes

Question 4

whats included in First generation sequencing?

Answer 4

• Chain-termination method - Chemical cleavage method

Question 5

what is second generation sequencing now also known as?

Answer 5

massively parallel sequencing (MPS)

Question 6

whats included in second generation sequencing?

Answer 6

 Pyrosequencing
 Ion torrent
 Reversible terminator sequencing
 SOLiD sequencing by ligation

Question 7

Third and fourth generation DNA sequencing uses what?

Answer 7

unamplified DNA.

Question 8

who and when was Chain-termination method discovered?

Answer 8

Fred Sanger in the 1970s – Sanger sequencing.

Question 9

what does polyacrylamide gel electrophoresis do?

Answer 9

fragments that differ by a single nucleotide length can be separated.

Question 10

what are the components of Chain-termination method?

Answer 10

• DNA molecule to be sequenced (target)
• Oligonucleotide primer to target the internal starting point
• DNA polymerase for extension
• 4 Deoxyribonuceloside triphosphates (dNTPs – dATP, dTTP, dCTP and dGTP.)

Question 11

A small amount of what is added during the Chain-termination method?

Answer 11

dideoxynucleoside triphosphates are added (ddNTPs)

Question 12

whats the difference between ddNTP compared to a dNTP?

Answer 12

sugar is dideoxyribose in ddNTP rather than deoxyribose.

Question 13

With dideoxyribose the 3ʹ carbon lacks what?

Answer 13

the hydroxyl group.

Question 14

How does a ddNTP block further elongation?

Answer 14

lack of a free hydroxyl group a connection cannot be formed with the next nucleotide

Question 15

what are the first three steps of Chain-termination method?

Answer 15

• DNA is denatured to produce single stranded DNA.
• Oligonucleotide primers anneal and with DNA polymerase chain extension
occurs with dNTP building blocks.
• ddNTPs are also added that can be labelled according to the type of base.

Question 16

what are the 4th, 5th and 6th steps of Chain-termination method?

Answer 16

• chain termination occurs.
• These products can then be separated by size using electrophoresis.
• For CE The fragments are detected using a fluorescent detector that can discriminate each labelled ddNTP.

Question 17

in Chain-termination method, what does the strands 3’ end have?

Answer 17

common 5ʹ end but the 3ʹ end will be variable and differ by a single nucleotide length.

Question 18

In polyacrylamide gel electrophoresis where will you find shorter fragments?

Answer 18

near top of the gel

Question 19

only ___ddNTP per tube.

Answer 19

one

Question 20

Capillary Electrophoresis can be done in?

Answer 20

a single tube

Question 21

In Capillary Electrophoresis what happens to dNTPs?

Answer 21

they are labelled with a fluorescent marker.

Question 22

in Capillary Electrophoresis what happens with a laser?

Answer 22

A laser excites the fluorophores and the fluorescent

signal is recorded. can be read.

Question 23

in Chain-termination method how many strands are copied ?

Answer 23

Only one strand is copied unlike PCR.

Question 24

where is the Purified DNA template for sequencing found?

Answer 24

obtained from molecular cloning or PCR.

Question 25

If using a PCR product for sequencing what primers can be used?

Answer 25

the same PCR primers | can be used or an internal primer

Question 26

why do genomes need to be sequenced in sections and multiple times ?

Answer 26

because o the limit this needs to be done to identify errors.

Question 27

what needs to happened to the shorter sequenced fragments for the larger genome sequence to be determined?

Answer 27

need to be assembled in the correct order

Question 28

what is the shotgun method?

Answer 28

sequenced fragments can be examined for overlapping sequencing to assemble

Question 29

what genomes have been assembled by this de novo sequencing shot-gun approach ?

Answer 29

prokaryotic smaller genomes

Question 30

what is Resequencing ?

Answer 30

A less computer intensive approach is Resequencing where a reference genome from the same species or similar species (de novo) is used.

Question 31

why are multiple reads done?

Answer 31

to deduce sequence and errors

Question 32

To ensure that errors are identified at least at least two many fold sequence depth or coverage is required?

Answer 32

5

Question 33

what can help deduce sequence errors?

Answer 33

Using a reference genome can help with assembly, making it quicker and more accurate.

Question 34

when was the first draft of the human genome project completed?

Answer 34

2001

Question 35

when was the first finished sequence ?

Answer 35

2003

Question 36

why is each region sequenced multiple times?

Answer 36

to enable an accurate sequence | identifies error in individual sequence reads.

Question 37

studying human genomes would be _____ and ______ if using this chain termination method.

Answer 37

slow | costly

Question 38

how long was chain termination the standard sequencing method?

Answer 38

30 years

Question 39

what was the main limitation of chain sequencing method?

Answer 39

sequencing throughput of the this method

Question 40

when was next generation sequencing introduced?

Answer 40

2005 onwards

Question 41

what is NGS also known as ?

Answer 41

massively-parallel DNA sequencing (MPS)

Question 42

what does NG enable us to do ?

Answer 42

enabled thousands or millions of DNA fragments to be sequenced in parallel in a single experiment reducing the cost.

Question 43

Whats good about NGS?

Answer 43

individual reads are much shorter but the overall throughput is much greater.

Question 44

whats the main difference between chain termination and massively parallel DNA sequencing?

Answer 44

MPS can sequence millions of DNA fragments simultaneously.

Question 45

MPS platforms fit into two broad categories which are?

Answer 45

1. Sequencing of PCR products, sequencing fragments of 35 – 800 nucleotides (nt) with a sequencing throughput of 1000 to a million Mb of DNA. 2. Sequencing of unamplified single-molecule sequencing, sequencing fragments thousands of nucleotides long but with a lower sequencing throughput of 100-1000 Mb.

Question 46

Next gen sequencing workflow?

Answer 46

1. extract DNA from patient sample 2. sharing of DNA with a method of choice + end rapid fragments 3. ligation of specific adaptors+ ligation of barcodes for multiplex beads 4. library selection and purification using magnetic streptavidin beads 5. amplify using PCR

Question 47

After the DNA is broken down by sonication to small fragments lengths then what happens?

Answer 47

it is then immobilised to create a DNA library

Question 48

How is a DNA library created for NGS ?

Answer 48

1. using oligonucleotides and adaptors bound to a solid glass support 2. Or the use of metallic beads coated with the protein streptavidin (SA) with a biotin labelled adaptor.

Question 49

What is the final step for preparation of sequence library?

Answer 49

PCR amplification of the immobilised DNA fragments to produce a sufficient number of copies to be sequenced.

Question 50

IMMOBILISATION OF DNA FRAGMENTS GLASS

Answer 50

DO IT