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MOLECULAR BIOLOGY > PREFI LEC: DNA SEQUENCING > Flashcards

PREFI LEC: DNA SEQUENCING Flashcards

1
Q

Refers to the order of the nucleotides in the DNA molecule

A

DNA SEQUENCE

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2
Q

Applications of DNA sequencing in medical laboratory:
1. Detection of mutation 2. Typing microorganisms 3. Identifying human haplotypes 4. Designating polymorphisms 5. Treatment strategies

A

DNA SEQUENCE

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3
Q

Sequencing Methods:

A
  1. Direct sequencing: manual and automated
  2. Pyrosequencing
  3. Bisulfite DNA sequencing
  4. RNA sequencing
  5. Next-Generation sequencing
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4
Q

Direct determination of the order, or sequence of nucleotides in a DNA polymer

A

DIRECT SEQUENCING

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5
Q

Most specific & direct method for identifying genetic lesions (mutations)/ polymorphisms

A

DIRECT SEQUENCING

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6
Q

2 TYPES OF DIRECT SEQUENCING

A
  1. Manual sequencing (chemical/ MaxamGilbert & Sanger sequencing)
  2. Automated fluorescent sequencing (dye primer & dye terminator sequencing)
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7
Q

2 PROCESS IN MANUAL DNA SEQUENCING

A
  1. Chemical (Maxam-Gilbert) Sequencing
  2. Dideoxy Chain Termination (Sanger) Sequencing
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8
Q

 Requires a ds/ss version of the DNA region to be sequenced, with 1 end radioactively labeled (32P)

A
  1. Chemical (Maxam-Gilbert) Sequencing
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9
Q

 Allan M. Maxam & Walter Gilbert

A

Chemical (Maxam-Gilbert) Sequencing

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10
Q

Sequencing proceeds in 4 separate reactions

A

Chemical (Maxam-Gilbert) Sequencing

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11
Q

Template used in Chemical (Maxam-Gilbert) Sequencing

A

labeled fragment

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12
Q

Addition of a ______ = ssDNA would break at specific nucleotides

A

strong reducing agent (10% piperidine)

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13
Q

After reactions: fragments will be separated by size on a ______

A

denaturing polyacrylamide gel (6-20%)

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14
Q

Short fragments (up to 50bp) =

A

1-2 hours

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15
Q

Long fragments (>150 bp) =

A

7-8 hours

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16
Q

 Frederick Sanger
 Uses dideoxynucleotides (ddNTPs) to determine the order/sequence of nucleotides in a nucleic acid
 Primer complementary to DNA to be sequenced

A

Dideoxy Chain Termination (Sanger) Sequencing

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17
Q

Product detection of sequencing of Dideoxy Chain Termination (Sanger) Sequencing

A
  1. Primer is attached at the 5’ end to a 32P/fluorescent dye-labeled nucleotide
  2. Incorporate 32P/35S-labeled dNTPs in the nucleotide sequencing reaction mix (internal labeling)
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18
Q

are added, terminating the DNA synthesis (chain termination)

A

ddNTPs

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19
Q

5’-3’ phosphodiester bond cannot be established to incorporate a subsequent nucleotide

A

Lack OH

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20
Q

Components: Mixed in 4 reaction tubes

A
  1. DNA template (PCR product)
  2. Radioactively-labeled primer
  3. Enzyme (DNA polymerase)
  4. dNTPs (all 4)
  5. Buffer (20 mM EDTA, formamide, gel tracking/loading dyes)
  6. Different ddNTPs in each of the 4 tubes
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21
Q

Sequencing reaction of Dideoxy Chain Termination (Sanger) Sequencing

A

thermal cycler (cycler sequencing)

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22
Q

Automated reading of DNA sequence ladder requires fluorescent dyes (4 distinct colors) to label primers / sequencing events
1. Fluorescein
2. Rhodamine
3. Bodipy (4,4-difluoro-4-bora-3a,4a-diazas-indacene)

A

AUTOMATED FLUORESCENT SEQUENCING

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23
Q

Fluorescent dyes can be distinguished by

A

automated sequencers

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24
Q

Approaches (to label fragments according to their terminal ddNTP):

A

dye primer & dye terminator sequencing

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25
Q

Fragments ending in ddATP, read as A in the sequence =

A

green dye

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26
Q

Fragments ending in ddCTP, read as C in the sequence =

A

blue dye

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27
Q

Fragments ending in ddGTP, read as G in the sequence =

A

black/yellow dye

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28
Q

Fragments ending in ddTTP, read as C in the sequence =

A

red dye

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29
Q

4 different fluorescent dyes are attached to 4 separate aliquots of the sample

A

Dye Primer Sequencing

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30
Q

Dye molecules are attached to the 5’ end of the primer = 4 versions of the same primer w/ different dye labels

A

Dye Primer Sequencing

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31
Q

Products are labeled at the 5’ end using the dye color associated w/ the ddNTP at the end of the fragment

A

Dye Primer Sequencing

32
Q

1 of the 4 fluorescent dyes attached to each of the ddNTPs

A

Dye Terminator Sequencing

33
Q

All 4 sequencing reactions are performed in the same tube

A

Dye Terminator Sequencing

34
Q

Product fragments are labeled at the 3’ end

A

Dye Terminator Sequencing

35
Q

 4 sets of sequencing products in each reaction are loaded onto a single gel lane/capillary
 Fluorescent dye colors distinguish which nucleotide is at the end of each fragment
 Fluorescent detection equipment yields results as electropherogram
 Base calling: process of bases ID in a sequence by sequencing software  If not clear, N will replace A, C, T, or G

A

Automated Electrophoresis

36
Q

Determines a DNA sequence without having to make a sequencing ladder

A

PYROSEQUENCING

37
Q

 Relies on the generation of light (luminescence) when nucleotides are added to a growing DNA strand
 No gels, fluorescent dyes, ddNTPs

A

PYROSEQUENCING

38
Q

Reaction mix components PYROSEQUENCING

A
  1. ssDNA template 2. Sequencing prime 3. Sulfurylase 4. Luciferase 5. Substrates: adenosine-5’-phosphosulfate (APS) & luciferin
  2. 1 of the 4 dNTPs is added to predetermined order of the reaction
39
Q

A.K.A. methylation-specific sequencing

A

BISULFITE DNA SEQUENCING

40
Q

 Chain termination sequencing designed to detect methylated cytosine nucleotides
 2-4 µg of genomic DNA is cut with restriction enzymes to facilitate denaturation

A

BISULFITE DNA SEQUENCING

41
Q

DNA is denatured (97ºC for 5 mins) & exposed to bisulfate solution (sodium bisulfite, NaOH, hydroquinone) for 16-20 hrs
 Cytosines are deaminated  uracil  5-methylcytosines are unchanged  Can be detected by Sanger sequencing/ pyrosequencing

A

BISULFITE DNA SEQUENCING

42
Q

 Early approaches: used RNase to cut endlabeled RNA at specific nucleotides
 Other approaches:
 based on amino acid sequence  based on sequencing of its complementary DNA

A

RNA SEQUENCING

43
Q

 Based on single-molecule sequencing technology & virtual terminator nucleotides  mRNA is captured by immobilized polydT oligomers (through their polyA tails)
 RNA w/o polyA tails: initial treatment w/ polyA polymerase
 4 reversibly dye-labeled nucleotides are sequentially added

A

DIRECT RNA SEQUENCING

44
Q

A.K.A. massive parallel sequencing

A

NEXT-GENERATION SEQUENCING (NGS)

45
Q

 Designed to sequence large numbers of templates carrying millions of bases
 Powerful computer data assembly systems (bioinformatics, computer software and support) are required

A

NEXT-GENERATION SEQUENCING (NGS)

46
Q

Require the preparation of a sequencing library (sets of DNA fragments representing the regions to be sequenced)

A

NEXT-GENERATION SEQUENCING (NGS)

47
Q

 Collection of genes that have been grouped for testing, enabling simultaneous sequencing of all genes (2 to >1000 genes)
 Focuses on targeted selection of specific genes for a specific purpose

A

Gene Panels

48
Q

3 TYPES OF Gene Panels

A

HOT SPOT PANEL
TARGETED PANEL
VERY LARGE PANEL

49
Q

target regions of specific genes known to affect treatment response, disease state, or clinical condition

A

“Hot-spot” panels

50
Q

critical genes in particular diseases (hematological-cancer specific, solidtumor specific)

A

Targeted panels

51
Q

diagnostic, prognostic, discovery purposes

A

Very large panels (>3000 genes)

52
Q

 Collection of DNA fragments (100-1000 bp) to be sequenced
 Represents a broad, comprehensive collection of DNA sequences (e.g., genomic or cDNA), allowing for genome-wide/large-scale analyses

A

NGS Library

53
Q

synthetic short dsDNA carrying sequences complementary to a single primer pair, which may contain short sequences that will ID the sample (indexing / bar coding)

A

Adapters

54
Q

 The regions to be sequenced are enriched by:
1. Probe hybridization
 Probes = biotinylated oligonucleotides complementary to specific gene regions

A

Targeted Libraries

55
Q

loss of library fragments from the sequenced regions

A

Allele dropout

56
Q

4 NGS Platforms

A
  1. Ion-conductance
  2. Reversible dye terminator sequencing
  3. Sequencing by ligation
  4. Nanopore sequencing
57
Q

Indexed libraries (gene panels) are amplified using primers immobilized on microparticles (beads) in aqueous oil emulsion using adapters on the library fragments complementary to the immobilized primers

A

Ion-Conductance

58
Q

 Captured/amplified fragments are hybridized to immobilized primers on a solid surface (flow cell)
 Labeled nucleotides are applied to the flow cell & incorporated into growing chains by DNA polymerase at each polony location

A

Reversible Dye Terminator Sequencing

59
Q

Uses a pool of labeled oligonucleotide DNA ligase to identify the template sequence through the known probe sequences

A

Sequencing by Ligation

60
Q

 Does not require fragmentation & amplification of the template DNA
 Each nucleotide can be identified by a disruption in current as it passes through the pore
 Also used for direct RNA sequencing

A

Nanopore Sequencing

61
Q

 Optical signals are translated to a nucleotide sequence (base calling), which is measured by the Phred score, acceptable = 2-3 (100-1000-fold certainty of a correct call)
 Each sequence is compared to a reference sequence (“normal”) through read alignment

A

Data Analysis

62
Q

based on comparison w/ the reference sequence (SNVs, indels, rearrangement sequences, CNVs)

A

Variant ID

63
Q

Sequence variations from the reference are arranged in a

A

variant call file (VCF)

64
Q

performed for critical variants ID

A

Annotations

65
Q

t or f

Confidence in the variant call is determined by sequence quality & coverage = at least 500x (recommended)

A

t

66
Q

Involves using computer technology (in silico) to collect, store, analyze, & disseminate biological data & information (computational biology)

A

BIOINFORMATICS

67
Q

 System for homology searches
 Searches GenBank  Searches can be made of NA & amino acid sequences
 Limits & parameters can be added (type of organisms)
 Matches/hits = diagram showing alignments & color code

A

BASIC LOCAL ALIGNMENT SEARCH
TOOL (BLAST)

68
Q

Assigned a universal nomenclature for mixed, degenerate, or wobble bases

A

International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular
Biology (IUB)

69
Q

Assigned a universal nomenclature for mixed, degenerate, or wobble bases

A

International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular
Biology (IUB)

70
Q

to decipher the sequence of the complete human genetic material (entire genome), identify all genes contained within the genome, & provide research tools to analyze all this genetic information

A

THE HUMAN GENOME PROJECT (HGP) primary mission

71
Q

THE HUMAN GENOME PROJECT (HGP) established and headed by??

A

National Institutes of Health (NIH) headed by James Watson

72
Q

1st complete genome sequence (1984)

A

Epstein-Barr virus

73
Q

_____ (Institute Genomic Research) completed the:
 1st sequence of a free-living organism (Haemophilus influenzae)
 Sequence of the smallest free-living organism (Mycoplasma genitalium)

A

Craig Venter & colleague

74
Q

1st sequence of a free-living organism

A

Haemophilus influenzae

75
Q

Sequence of the smallest free-living organism

A

Mycoplasma genitalium

MOLECULAR BIOLOGY (9 decks)

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