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Molecular Biology Lecture > 3: DNA Sequencing Techniques (MIDTERMS) > Flashcards

3: DNA Sequencing Techniques (MIDTERMS) Flashcards

1
Q

These two methods in sequencing DNA became the basis of more sophisticated DNA techniques

A

Sanger and Maxam-Gilbert Technique

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2
Q

What are the three DNA sequencing discussed?

A
  1. Sanger Technique
  2. Maxam-Gilbert Technique
  3. Next Generation Technique
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3
Q

Technique made during the mid 1970’s

a. Sanger Technique
b. Maxam-Gilbert Technqiue
c. Both
d. NOTA

A

c. Both

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4
Q

Process of determining sequence of nucleotide bases

A

DNA Sequencing

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5
Q

How are nucleotides identified?

A

Based on the bases of their structures

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6
Q

Use this card to familiarize the process of DNA sequencing

A
  1. Break down DNA to smaller pieces
  2. The smaller pieces will be sequenced one at a time
  3. That portion of DNA will assemble the sequence into a single continuous order of nucleotide
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7
Q

T or F

Portion of DNA will assemble the sequence into a single continuous order of nucleotide to produce a sequence of genome

A

T

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8
Q

Conventional methods

a. Sanger Technique
b. Maxam-Gilbert Technqiue
c. Both
d. NOTA

A

c. Both

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9
Q

Sanger and Maxam-Gilbert Technique are the principle of what new sequencing method?

a. Robotics
b. Automated Sequencing
c. Both
d. NOTA

A

c. Both

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10
Q

Chain termination sequencing

a. Sanger Technique
b. Maxam-Gilbert Technqiue
c. Both
d. NOTA

A

a. Sanger Technique

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11
Q

What DNA sequencing technique:

→ Target DNA is copied many times
→ PCR-based techniques

A

Sanger Technique

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12
Q

T or F

Sanger Technique makes fragments of different lengths

A

T

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13
Q

→ 1991-2003

→ Process wherein the entire human genome or makeup of a human being is sequenced

A

Human Genome Project

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14
Q

When was the human genome project started and ended?

A

1991-2003

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15
Q

Technique wherein results are produced within days

a. Sanger Technique
b. Maxam-Gilbert Technique
c. Both
d. NOTA

A

d. NOTA (next generation sequencing technique yarn)

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16
Q

Uses fluorescence

a. Sanger Technique
b. Maxam-Gilbert Technique
c. Next generation technique
d. ALL of the above
e. NOTA

A

d. ALL of the above

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17
Q

What DNA sequencing technique:

→ PCR-based method

→ Modified DNA replication method

→ Growing chains are terminated by dideoxynucleotides/ddNTPs

A

Sanger Technique: chain termination method

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18
Q

What are the five requirements in Sanger Technique?

A
  1. DNA primer
  2. DNA polymerase
  3. dNTP
  4. four ddNTPs
  5. Template DNA
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19
Q

Sanger Technique:

What is the 1st step?

A

DNA Denaturation

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20
Q

Sanger Technique:

What is the 2nd step after denaturation?

A

Aliquot to 4 tubes

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21
Q

Sanger Technique:

What is the 3rd step after aliquoting to 4 tubes

A

Incorporate reagents: Primer, dNTP, DNA Polymerase

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22
Q

Sanger Technique:

What are the reagents added before ddNTPs?

A

Template ssDNA, Primer, dNTP, DNA Polymerase

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23
Q

Sanger Technique:

After incorporating reagents, what should be added to the 4 tubes?

A

Four dideoxynucleotides or ddNTPs in each tube

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24
Q

Sanger Technique:

T or F

In adding ddNTPS in tube, 2 ddNTPS should be added per tube

A

F (1 specific ddNTP should be added per tube; example: Tube 1 - ddATP → Tube 2 - ddTTP)

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25
Q

Sanger Technique:

What is the last step after adding ddNTP?

A

Gel electrophoresis

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26
Q

How is the gel electrophoresis product of Sanger technique read?

A

BOTTOM to TOP and individually (tama nman khit sa kama rin eme)

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27
Q

Sanger Technique:

In gel electrophoresis, DNA fragments move from?

A

negative to positive pole

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28
Q

Sanger Technique:

T or F

DNA fragments move from negative to positive pole because of the base backbone

A

F (PHOSPHATE backbone which is negative charged)

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29
Q

Sanger Technique: Identify what size of fragment

→ Migrate at the bottom
→ lighter
→ Read first

A

Shorter fragments

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30
Q

Sanger Technique: Identify what size of fragment

→ Migrate at the top
→ Heavier

A

Longer fragments

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31
Q

Sanger Technique:

Migrate at the bottom and is read first

a. Longer fragments
b. Shorter fragments
c. both
d. NOTA

A

b. Shorter fragments

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32
Q

Sanger Technique:

After gel electrophoresis, what is the product formed?

A

Complementary strand

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33
Q

→ Has OH on 3’ carbon
→ Allows attachment of nucleotides
→ Has more concentration

A

dNTP

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34
Q

→ Lacks OH on the 3’ carbon
→ Terminates synthesis of new strands
→ Fluoresce
→ Added in chain randomly
→ complementary to target DNA

A

ddNTP

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35
Q

Allows attachment of nucleotides

a. dNTP
b. deoxynucleotide triphosphate
c. both
d. NOTA

36
Q

Has OH on 3’ carbon

a. ddNTP
b. dideoxynucleotide triphosphate
c. both
d. NOTA

A

d. NOTA (dNTP/deoxynucleotide triphosphate)

37
Q

Terminates synthesis of new strands and fluoresce

a. ddNTP
b. dNTP
c. both
d. NOTA

38
Q

Added in chain randomly and complementary to target DNA

a. dNTP
b. dideoxynucleotide triphosphate
c. both
d. NOTA

A

b. dideoxynucleotide triphosphate

39
Q

T OR F

ddNTP has more concentration than dNTP

A

F (reverse; dNTP pa rin lamang sa concenctration)

40
Q

→ DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases

→ Chemicals are used that selectively attack purines and pyrimidines

A

Maxam-GIlbert Technique: Chemical Sequencing

41
Q

Use this card to familiarize the process of Maxam-Gilbert Technique

A
  1. Denature DNA
  2. Chemical modification of DNA/Labelling
  3. Purification of the DNA fragment to be sequenced
  4. Chemical treatment generates breaks in DNA
  5. Addition of Piperidine
  6. Gel electrophoresis
42
Q

Maxam-GIlbert Technique:

What is the 1st step

A

Denature the DNA

43
Q

What is the temperature for denaturing DNA?

A

95 degree celcius

44
Q

Maxam-GIlbert Technique:

After denaturing DNA, what is the next step?

A

Chemical modification aka radioactive labeling at one 5’ end of the DNA by kinase reaction using gamma -32 P ATP

45
Q

Maxam-GIlbert Technique:

In labelling the strand, what should be used?

A

gamma -32 P ATP

46
Q

Maxam-GIlbert Technique:

T or F

Radioactive labeling at one 3’ end of the DNA by kinase reaction using gamma -32 P ATP

A

F (Radioactive labeling at one 5’ END of the DNA by kinase reaction using gamma -32 P ATP)

47
Q

Maxam-GIlbert Technique:

T or F

Purpose of radioactive labelling is to mark the strand before cutting so that there are three 5’end

A

F (mark the strand before cutting so that there are TWO 5’end)

48
Q

Maxam-GIlbert Technique:

After radioactive labelling, what is the 3rd step?

A

Purification of the DNA fragment to be sequenced

49
Q

Maxam-GIlbert Technique:

After purification, what is the next step?

A

Chemical treatment generates breaks in DNA

50
Q

Maxam-GIlbert Technique:

What is the Chemical Process, Nucleic Acid Modifier, and Minutes Reaction Time for the base C?

A

Maxam-GIlbert Technique:

Chemical Process: Splits C rings
Nucleic Modifier: Hydrazine + Salt
Reaction Time: 8 mins

51
Q

Maxam-GIlbert Technique:

What is the Chemical Process, Nucleic Acid Modifier, and Minutes Reaction Time for the base T + C?

A

Chemical Process: Splits pyrimidine rings
Nucleic Modifier: Hydrazine
Reaction Time: 8 mins

52
Q

Maxam-GIlbert Technique:

What is the Chemical Process, Nucleic Acid Modifier, and Minutes Reaction Time for the base G + A?

A

Chemical Process: Protonates purines
Nucleic Modifier: Formic acid
Reaction Time: 5 mins

53
Q

Maxam-GIlbert Technique:

What is the Chemical Process, Nucleic Acid Modifier, and Minutes Reaction Time for the base G?

A

Chemical Process: Methylates G
Nucleic Modifier: Dimethylsulphate
Reaction Time: 4 mins

54
Q

Maxam-GIlbert Technique:

Hydrazine + salt

a. C
b. T + C
c. both
e. NOTA

55
Q

Maxam-GIlbert Technique:

Hydrazine

a. G + A
b. G
c. both
e. NOTA

A

e. NOTA (T + C or PYRIMIDINES)

56
Q

Maxam-GIlbert Technique:

Formic Acid

a. G + A
b. G
c. both
e. NOTA

57
Q

Maxam-GIlbert Technique:

Dimethylsulphate

a. G + A
b. G
c. both
e. NOTA

58
Q

Maxam-GIlbert Technique:

Protonates

a. G + A
b. G
c. both
e. NOTA

59
Q

Maxam-GIlbert Technique:

Methylation

a. G + A
b. G
c. both
e. NOTA

60
Q

Maxam-Gilbert Technique:

Splits rings

a. C
b. T + C
c. both
e. NOTA

61
Q

Maxam-GIlbert Technique:

Splits C rings

a. Hydrazine
b. Hydrazine + salt
c. both
e. NOTA

62
Q

Maxam-GIlbert Technique:

Splits purine rings

a. T + C
b. C
c. both
e. NOTA

A

e. NOTA (there’s no splitting of purine only PYRIMIDINE by using hydrazine)

63
Q

Maxam-GIlbert Technique:

Minutes reaction time: 8 minutes

a. T + C
b. C
c. both
e. NOTA

64
Q

Maxam-GIlbert Technique:

After treating with chemical, what is the next step?

A

Addition of Piperidine

65
Q

Maxam-GIlbert Technique:

This cuts sequence of the nucleic acid modifier

A

Piperidine

66
Q

Maxam-GIlbert Technique:

After addition of Piperidine, what is the last step?

A

Gel electrophoresis

67
Q

Maxam-GIlbert Technique:

Gel electrophoresis of Maxam-Gilbert Technique is read from?

A

BOTTOM to TOP, by PAIR

68
Q

Read from bottom to top

a. Sanger Technique
b. Maxam-Gilbert Technique
c. both
d. NOTA

69
Q

→ also called massively parallel sequencing

→ methods in which millions of DNA templates are sequenced simultaneously in a single reaction.

A

Next Generation Sequencing

70
Q

Next Generation Sequencing:

nucleotide sequence data is then assembled using ?

A

computer algorithms into a continuous genome sequence.

71
Q

Next Generation Sequencing:

What is the 1st step?

A

Ligation: fragments of cellular DNA are ligated to oligonucleotides referred to as adapters.

72
Q

Next Generation Sequencing:

What are the fragments of DNA ligated to oligonucleotides called?

73
Q

Next Generation Sequencing:

After ligation with adapters, what is next step?

A

Denaturation and strands are captured on solid surface

74
Q

Next Generation Sequencing:

Which serves as primer for DNA polymerase?

A

annealed oligonucleotide

75
Q

Next Generation Sequencing:

T or F

After DNA extension, two long strands separate, leaving 1 strand attached by a covalent bond to a fixed oligonucleotide.

76
Q

Next Generation Sequencing:

Can form a bridge with another fixed oligonucleotide in another round of PCR proceeds.

A

DNA fragment

77
Q

Next Generation Sequencing:

After multiple rounds, what forms in the location of each original cellular fragment

A

a small patch of DNA

78
Q

Next Generation Sequencing:

T or F

These single-stranded DNA molecules represent two sequences that are complementary to each other

79
Q

Next Generation Sequencing:

T or F

Nucleotides contain a starting group on their 3’ ends

A

F (Nucleotides contain a BLOCKING group on their 3’ ends)

80
Q

Next Generation Sequencing:

induces the nucleotide to fluoresce and the color is recorded

81
Q

Next Generation Sequencing:

T or F

The cycle can be repeated up to 20x only

A

F (repeated up to 100 X)

82
Q

Next Generation Sequencing:

assemble overlapping sequence fragments into longer pieces and in this way determine the overall sequence of a genome

A

Software packages

83
Q

IF YOU SEE THIS CARD, PAKIREVIEW ANG STEPS NG NEXT GEN SEQUENCING PLS AND HIRAP NIYA GAWAN NG CARD FR

A

DALI NA PLS BB : (((

84
Q

Denaturation Step

a. Sanger
b. Maxam-GIlbert
c. Next Generation sequencing
d. ALL
e. NOTA

85
Q

DNA Amplification and Extension
a. Sanger
b. Maxam-Gilbert
d. ALL
e. NOTA

86
Q

Ligation

a. Sanger
b. Maxam-GIlbert
c. Next Generation sequencing
d. ALL
e. NOTA

A

c. Next Generation sequencing

Molecular Biology Lecture (17 decks)

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