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Molbio. > Sequencing methods > Flashcards

Sequencing methods Flashcards

1
Q

DNA sequencing is the process of determining the —

enumerate the nucleotides

A

order of nucleotides

adenine, thymine, guanine, cytosine

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2
Q

what are the two main types of DNA sequencing?

A
  • Sanger sequencing
  • next-generation sequencing
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3
Q

used to terminate DNA synthesis in Sanger sequencing

A

dideoxynucleotides (ddNTPs)

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4
Q

chain-termination method that uses ddNTPs to terminate DNA synthesis

A

Sanger sequencing

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5
Q

dideoxynucleotides lack — which necessary for DNA synthesis to continue

A

3’-hydroxyl group

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6
Q

how to perform sanger sequencing? enumerate.

A
  1. DNA sample is amplified (PCR)
  2. amplified DNA is incubated with a mixture of ddNTPs and dNTPs
  3. ddNTPs will randomly terminate DNA synthesis, resulting in a series of DNA fragments of different lengths.
  4. DNA fragments are separated by gel electrophoresis.
  5. The order of the nucleotides in the DNA molecules is determined by reading the order of the bands on the gel.
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7
Q

ddNTPs will randomly terminate DNA synthesis, what is the product?

A

series of DNA fragments of different lengths

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8
Q

DNA fragments are then separated by — and the sequence of the DNA molecule is dtermined by?

A

separated by gel electrophoresis

sequence of DNA molecule is determined by reading the order of the bands on the gel

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9
Q

collection of methods that can sequence DNA much faster and more cheaply.

A

next-generation sequencing

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10
Q

this involve massively parallelizing the sequencing process

A

next-generation sequencing

DNA molecules are sequenced simultaneously

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11
Q

DNA molecules are sequenced simultaneously. This is achieved by using a variety of techniques such as –

A
  • sequencing by synthesis
  • sequencing by ligation
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12
Q

true or false

SBS can sequence entire genomes at a relatively low cost.

A

FALSE
next-generation sequencing

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13
Q

NGS is used for —

A
  • research
  • diagnostics
  • personalized medicine
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14
Q

first DNA sequencing method and is widely used today. give the advantage and disadvantages of this method.

A

SANGER METHOD

advantages: simple and straightforward

disadvantage: time consuming and expensive to sequence large DNA molecules

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15
Q

advantages of NGS

A
  • can sequence large DNA molecules
  • fast and cheap
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16
Q

most common type of NGS method, it works by sequencing the DNA molecule on one nucleotide at a time.

A

sequencing by synthesis

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17
Q

DNA molecule is first attached to a solid surface. then a mixture of nucleotides is added to the surface. the nucleotides will bind to the complementary nucleotides in the DNA molecule. this method refers to?

A

sequencing by synthesis

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18
Q

used to image the surface and determine the order of the nucleotides in the DNA molecules. this method refers to?

A

LASER

sequencing by synthesis

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19
Q

sequencing the DNA molecule one fragment at a time.

A

sequencing by ligation

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20
Q

DNA molecule is first fragmented into small pieces then the fragments are attached to adapters. the adapters contain sequences that are known to bind to the sequencing machine. the fragments are then ligated together to form a long chain. the sequencing machine then reads to the order of the nucleotides in the chain. this method refers to?

A

sequencing by ligation

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21
Q

lecture

enumerate the importance of DNA sequence information

A
  • detecting mutations
  • typing microorganisms
  • identify human haplotypes
  • designating polymorphisms
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22
Q

lecture

most specific esp when we use to identify genetic lesions, mutations, and polymorphisms

A

direct sequencing

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23
Q

lecture

enumerate the 2 manual sequencing methods of direct

A
  1. Chemical (Maxam-Gilbert) Sequencing
  2. Dideoxy Chain termination (Sanger sequencing)
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24
Q

lecture

the labeled fragment or template is aliqouted into 4 tubes and each of this tubes contain a base modifier, this refers to what method?

A

Chemical (Maxam-Gilbert) sequencing

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25
Q

lecture - manual sequencing

a reducing agent which breaks the ssDNA at particular specific nucleotide of each base in each tube

A

10% pteridine

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26
Q

lecture

enumerate the base modifiers and at what chain breaks at —

A
  1. dimetlysulphate = G
  2. formic acid = G + A
  3. hydrazine = T + C
  4. hydrazine + salt = C
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27
Q

lecture

this base modifier methylates G
chain breaks at?

time (min @ 25C)?

A

Dimethylsulphate
breaks at G

4 @25 C

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28
Q

lecture

this base modifier protonates purine and chain breaks at G+A

A

formic acid

5 at 25C

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29
Q

lecture

splits pyrimidine rings and chain breaks at T+C

A

hydrazine

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30
Q

lecture

this base modifier splits only C rings, chain breaks at C

A

hydrazine + salt

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31
Q

lecture

this method is very direct way to sequence DNA but **not practical for high throughput ** esp. for long fragments because this is tedious to do.

A

Chemical (Maxam-Gilbert) sequencing

32
Q

lecture

there is the addition of a nucleotide triphosphate - which requires a free hydroxyl group from 3’ carbon. this method refers to?

A

Dideoxy Chain termination
Sanger sequencing

33
Q

lecture - manual sequencing

strand synthesis reaction is catalyzed by a type of DNA polymerase known as?

A

Klenow fragment

34
Q

lecture

method of sanger sequencing

A
  1. anneal a short oligonucleotide (17-24 nucleotides), to the same position on each molecule.
  2. ddTTP is then added to stop the reaction.
  3. perform our DNA synthesis in 4 separate tubes
  4. perform electrophoresis under denatured condition
35
Q

lecture - sanger sequencing

act as a substrate that in the 4 tubes

A

4 dideoxynucleotides
4 deoxyribonucleotide triphosphates

36
Q

lecture

products of the four-sequencing reaction

A

sequencing ladder

37
Q

lecture

this is performed to visualize the DNA fragments which will appear as horizontal bands on an x-ray film.

A

AUTORADIOGRAPHY

it is performed bcos of radioactive ddATP is included in your reaction.

38
Q

lecture

sequencing enzyme which has low processivity means that it can only synthesize short DNA strands before dissociating from the template

A

Klenow polymerase

39
Q

lecture

drawback of klenow polymerase

A

limit the length of the sequence tjat can be obtained from a single experiment to only 250 base pairs

40
Q

lecture

modified version of the DNA polymerase that is encoded by bacteriophage T7

A

sequenase

to improve short synthesis problem

41
Q

lecture

this has high processivity that can synthesize longer strands compared to the klenow polymerase and NO EXONUCLEASE ACTIVITY making it ideal for chain termination sequencing

how many base pairs can be sequenced in just a single expirement?

A

sequenase

750 base pairs in just a single experiment

42
Q

lecture

use to inset our DNA and only good to use if your DNA fragments are shorter than 3kb

A

M13 vector

43
Q

lecture

this vector is needed to convert the double-stranded plasmid into a single-stranded form through the addition of an alkali or through boiling

A

PLASMID VECTOR
alternative for M13

44
Q

lecture

most common method for obtaining a template DNA for DNA sequencing

A

DENATURATION
adding of alkali | boiling

45
Q

lecture

a plasmid vector that contains an M13 origin of replication and which can be obtained as both a double- or single-stranded DNA version

this can be used — of DNA fragments

A

phagemid

more than 10 kb DNA fragments

46
Q

lecture

each of the dideoxynucleotide used in the reaction is labeled with a different fluoresent marker, this method refers to?

termination sequencing is only performed in one tube

A

automated method

47
Q

lecture - automated method

how can we separate the moleculres according to their lengths after the extension reaction?

A

polyacrylamide gel | capillary gel electrophoresis

48
Q

lecture

ratio of ddNTPs and dNTPs
this is important for the generation of a readable sequence

what is the effect if high concentration of ddNTPs or if it is too low?

A

1:1

  • high = polymerization will terminate to frequently early on the template
  • low = no termination that will occur
49
Q

lecture

the reactions are terminated by addition of a stop buffer, this contains —

A
  • 20 mM EDTA
  • formamide
  • gel loading dyes
50
Q

lecture

used to chelate cations and stop enzyme activity

A

20 mM EDTA

51
Q

lecture

this will denature the product of the synthesis reaction

A

formamide

52
Q

lecture

what are the gel loading dyes?

A
  • bromophenol blue
  • xylene cyanol
53
Q

lecture

first of the high throughput DNA sequencing or next generation sequencing technology. this is for large amount of data about genomic DNA

A

pyrosequencing

54
Q

lecture

can be automated in a massively parallel manner that neables hundreds of throusands of sequences that can be obtained at once or has even at a hundred million of base pairs in just a single run.

A

pyrosequencing

55
Q

lecture

the basis of — is the detection of pyrophosphate that is released during DNA synthesis. it requires preparation first of an identical single-stranded DNA molecule.

A

pyrosequencing

56
Q

lecture - pyrosequencing

pyrophosphate will then combine with adenosine-5’-phosphosulfate in the presence of the enzyme ATP sulfurylase to form ATP. this ATP would drive the conversion of — by the enzyme —

A

conversion of luciferen to oxyluciferin by the enzyme luciferase

57
Q

lecture

— will then generate light. the amount of light is detected after each cycle of nucleotide addition and the enzymatic reaction indicates the incorportaion of a complementary nucleotide.

A

oxyluciferin

58
Q

lecture

light is — to the amount of pyrophosphate produced and — to the number of nucleotides added

A

DIRECTLY PROPORTIONAL

59
Q

lecture

catalyzes the degregation of the excess dNTPs before we add next dNTP

A

apyrase

60
Q

lecture

advantages of pyrosequencing

A
  • rapid
  • can be automated
  • does not require electrophoresis
  • hundreds of thousands of sequences can be obtained
61
Q

lecture

sequence assembly can be done through?

A
  • shotgun approach
  • clone contig approach
62
Q

lecture

this sequence assembly will break down the genome randomly into short fragments. it will then be used to build-up through seeing which sequence will fit unto the length of the DNA

A

shotgun cloning approach

this is only done for smaller and shorter bacterial genome

63
Q

lecture

a presequencing phase during which a series of overlaping clones is identified. longer lengths which of overlaps twith each other.

overlapping clones are called?

A

clone contig approach

contig - overlapping clones

64
Q

lecture - shotgun cloning

if it ends in the 5’ single stranded end, — is used to fill in the 3’ recessed end of the complementary strand.

if it ends on the 3’ single stranded end, — is used to remove protruding end.

library - uniformly sized DNA fragments

A

5’ single stranded = polymerase
3’ = exonuclease

65
Q

lecture

vector used in shotgun cloning which will act as a sequencing template

A

E. coli

66
Q

lecture

this approach provides an accurate sequence of a large genome that contains repetitive DNA

what is the disadvantage?

A

clone contig approach

time consuming and requires effort

67
Q

lecture

enumerate the high capacity vectors

A
  1. bacterial artificial chromosome (BAC)
  2. yeast artificial chromosome (YAC)
68
Q

lecture - clone contig

each fragment is cloned in a vector and is sequenced from both ends to produce a sequence length of — base pairs

sequnce of both ends through the DNA fragment is called?

A

600 - 700 base pairs

pair end

69
Q

lecture

enumerate the techniques used in clone contig assembly (4)

A
  • chromosome walking
  • clone fingerprinting
  • interspersed repeat element PCR
  • sequence tagged site content analysis
70
Q

lecture

this technique is used to move relatively short distances along DNA molecules, using clone libraries prepared with lambda or cosmind vector

A

chromosome walking

71
Q

lecture

this is a technique where you are attempting to move short molecules through the vector

what could be the weakness for this technique?

aim: close one or more small gaps between contigs

A

CHROMOSOME WALKING

weakness: slow process can only assemble at aroung 15-20 clones

this is only attempted when the contig is for a short chromosome

72
Q

lecture

this is the identification of sequence features that are shared by a pair of clones. it also provide information on a physical structure of a cloned DNA fragment.

the simplest approach is to digest each clone with a variety of restriction endonucleases.

A

clone fingerprinting

73
Q

lecture

it uses primers that are design to anneal within repetitive DNA sequences and direct amplification of the DNA between adjacent repeats.

A

interspersed repeat element PCR (IRE-PCR)

74
Q

lecture

this can be used as a fingerprint in comparison to the other clones in order to identify potential overlaps.

A

DNA PCR

75
Q

lecture

this is when we search for pairs of clones that contain a specific DNA sequence that occurs at just one position in the genome under study.

A

sequence tagged site content analysis

76
Q

lecture

if two clones contain a specific DNA sequence that occurs at just one position in the genome must overlap. a sequence of this type is called?

A

sequence tagged site

77
Q

lecture

only requirement of sequence tagged site

A

it must occur at once only in the gene

Molbio. (6 decks)

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