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

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Flashcards in Lecture 3: Studying Human DNA: DNA sequencing (not finished) Deck (50)
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1

what san example of a sequence polymorphisms ?

single nucleotide polymorphisms (SNPs)

2

what does DNA sequencing determine?

DNA sequencing determine the precise order of nucleotides.

3

is there more than one method to sequence DNA?

yes

4

whats included in First generation sequencing?

- Chain-termination method - Chemical cleavage method

5

what is second generation sequencing now also known as?

massively parallel sequencing (MPS)

6

whats included in second generation sequencing?

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

7

Third and fourth generation DNA sequencing uses what?

unamplified DNA.

8

who and when was Chain-termination method discovered?

Fred Sanger in the 1970s – Sanger sequencing.

9

what does polyacrylamide gel electrophoresis do?

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

10

what are the components of Chain-termination method?

- 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.)

11

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

dideoxynucleoside triphosphates are added (ddNTPs)

12

whats the difference between ddNTP compared to a dNTP?

sugar is dideoxyribose in ddNTP rather than deoxyribose.

13

With dideoxyribose the 3ʹ carbon lacks what?

the hydroxyl group.

14

How does a ddNTP block further elongation?

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

15

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

• 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.

16

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

• 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.

17

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

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

18

In polyacrylamide gel electrophoresis where will you find shorter fragments?

near top of the gel

19

only ___ddNTP per tube.

one

20

Capillary Electrophoresis can be done in?

a single tube

21

In Capillary Electrophoresis what happens to dNTPs?

they are labelled with a fluorescent marker.

22

in Capillary Electrophoresis what happens with a laser?

A laser excites the fluorophores and the fluorescent
signal is recorded. can be read.

23

in Chain-termination method how many strands are copied ?

Only one strand is copied unlike PCR.

24

where is the Purified DNA template for sequencing found?

obtained from molecular cloning or PCR.

25

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

the same PCR primers
can be used or an internal primer

26

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

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

27

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

need to be assembled in the correct order

28

what is the shotgun method?

sequenced fragments can be examined for overlapping sequencing to assemble

29

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

prokaryotic smaller genomes

30

what is Resequencing ?

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