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2nd year sem 1: Forensic Genetics > Lecture 3: Studying Human DNA: DNA sequencing (not finished) > Flashcards

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

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1
Q

what san example of a sequence polymorphisms ?

A

single nucleotide polymorphisms (SNPs)

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2
Q

what does DNA sequencing determine?

A

DNA sequencing determine the precise order of nucleotides.

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3
Q

is there more than one method to sequence DNA?

A

yes

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4
Q

whats included in First generation sequencing?

A
  • Chain-termination method - Chemical cleavage method
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5
Q

what is second generation sequencing now also known as?

A

massively parallel sequencing (MPS)

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6
Q

whats included in second generation sequencing?

A

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

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7
Q

Third and fourth generation DNA sequencing uses what?

A

unamplified DNA.

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8
Q

who and when was Chain-termination method discovered?

A

Fred Sanger in the 1970s – Sanger sequencing.

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9
Q

what does polyacrylamide gel electrophoresis do?

A

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

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10
Q

what are the components of Chain-termination method?

A
  • 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.)
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11
Q

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

A

dideoxynucleoside triphosphates are added (ddNTPs)

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12
Q

whats the difference between ddNTP compared to a dNTP?

A

sugar is dideoxyribose in ddNTP rather than deoxyribose.

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13
Q

With dideoxyribose the 3ʹ carbon lacks what?

A

the hydroxyl group.

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14
Q

How does a ddNTP block further elongation?

A

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

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15
Q

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

A

• 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.

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16
Q

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

A
  • 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.
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17
Q

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

A

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

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18
Q

In polyacrylamide gel electrophoresis where will you find shorter fragments?

A

near top of the gel

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19
Q

only ___ddNTP per tube.

A

one

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20
Q

Capillary Electrophoresis can be done in?

A

a single tube

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21
Q

In Capillary Electrophoresis what happens to dNTPs?

A

they are labelled with a fluorescent marker.

22
Q

in Capillary Electrophoresis what happens with a laser?

A

A laser excites the fluorophores and the fluorescent

signal is recorded. can be read.

23
Q

in Chain-termination method how many strands are copied ?

A

Only one strand is copied unlike PCR.

24
Q

where is the Purified DNA template for sequencing found?

A

obtained from molecular cloning or PCR.

25
Q

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

A

the same PCR primers

can be used or an internal primer

26
Q

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

A

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

27
Q

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

A

need to be assembled in the correct order

28
Q

what is the shotgun method?

A

sequenced fragments can be examined for overlapping sequencing to assemble

29
Q

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

A

prokaryotic smaller genomes

30
Q

what is Resequencing ?

A

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

31
Q

why are multiple reads done?

A

to deduce sequence and errors

32
Q

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

A

5

33
Q

what can help deduce sequence errors?

A

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

34
Q

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

A

2001

35
Q

when was the first finished sequence ?

A

2003

36
Q

why is each region sequenced multiple times?

A

to enable an accurate sequence

identifies error in individual sequence reads.

37
Q

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

A

slow

costly

38
Q

how long was chain termination the standard sequencing method?

A

30 years

39
Q

what was the main limitation of chain sequencing method?

A

sequencing throughput of the this method

40
Q

when was next generation sequencing introduced?

A

2005 onwards

41
Q

what is NGS also known as ?

A

massively-parallel DNA sequencing (MPS)

42
Q

what does NG enable us to do ?

A

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

43
Q

Whats good about NGS?

A

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

44
Q

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

A

MPS can sequence millions of DNA fragments simultaneously.

45
Q

MPS platforms fit into two broad categories which are?

A
  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.
46
Q

Next gen sequencing workflow?

A
  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
47
Q

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

A

it is then immobilised to create a DNA library

48
Q

How is a DNA library created for NGS ?

A
  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.
49
Q

What is the final step for preparation of sequence library?

A

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

50
Q

IMMOBILISATION OF DNA FRAGMENTS GLASS

A

DO IT

2nd year sem 1: Forensic Genetics (8 decks)

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