PREFI LEC: DNA SEQUENCING Flashcards
1
Q
Refers to the order of the nucleotides in the DNA molecule
A
DNA SEQUENCE
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
3
Q
Sequencing Methods:
A
- Direct sequencing: manual and automated
- Pyrosequencing
- Bisulfite DNA sequencing
- RNA sequencing
- Next-Generation sequencing
4
Q
Direct determination of the order, or sequence of nucleotides in a DNA polymer
A
DIRECT SEQUENCING
5
Q
Most specific & direct method for identifying genetic lesions (mutations)/ polymorphisms
A
DIRECT SEQUENCING
6
Q
2 TYPES OF DIRECT SEQUENCING
A
- Manual sequencing (chemical/ MaxamGilbert & Sanger sequencing)
- Automated fluorescent sequencing (dye primer & dye terminator sequencing)
7
Q
2 PROCESS IN MANUAL DNA SEQUENCING
A
- Chemical (Maxam-Gilbert) Sequencing
- Dideoxy Chain Termination (Sanger) Sequencing
8
Q
Requires a ds/ss version of the DNA region to be sequenced, with 1 end radioactively labeled (32P)
A
- Chemical (Maxam-Gilbert) Sequencing
9
Q
Allan M. Maxam & Walter Gilbert
A
Chemical (Maxam-Gilbert) Sequencing
10
Q
Sequencing proceeds in 4 separate reactions
A
Chemical (Maxam-Gilbert) Sequencing
11
Q
Template used in Chemical (Maxam-Gilbert) Sequencing
A
labeled fragment
12
Q
Addition of a ______ = ssDNA would break at specific nucleotides
A
strong reducing agent (10% piperidine)
13
Q
After reactions: fragments will be separated by size on a ______
A
denaturing polyacrylamide gel (6-20%)
14
Q
Short fragments (up to 50bp) =
A
1-2 hours
15
Q
Long fragments (>150 bp) =
A
7-8 hours
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
17
Q
Product detection of sequencing of Dideoxy Chain Termination (Sanger) Sequencing
A
- Primer is attached at the 5’ end to a 32P/fluorescent dye-labeled nucleotide
- Incorporate 32P/35S-labeled dNTPs in the nucleotide sequencing reaction mix (internal labeling)
18
Q
are added, terminating the DNA synthesis (chain termination)
A
ddNTPs
19
Q
5’-3’ phosphodiester bond cannot be established to incorporate a subsequent nucleotide
A
Lack OH
20
Q
Components: Mixed in 4 reaction tubes
A
- DNA template (PCR product)
- Radioactively-labeled primer
- Enzyme (DNA polymerase)
- dNTPs (all 4)
- Buffer (20 mM EDTA, formamide, gel tracking/loading dyes)
- Different ddNTPs in each of the 4 tubes
21
Q
Sequencing reaction of Dideoxy Chain Termination (Sanger) Sequencing
A
thermal cycler (cycler sequencing)
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
23
Q
Fluorescent dyes can be distinguished by
A
automated sequencers
24
Q
Approaches (to label fragments according to their terminal ddNTP):
A
dye primer & dye terminator sequencing
25
Fragments ending in ddATP, read as A in the sequence =
green dye
26
Fragments ending in ddCTP, read as C in the sequence =
blue dye
27
Fragments ending in ddGTP, read as G in the sequence =
black/yellow dye
28
Fragments ending in ddTTP, read as C in the sequence =
red dye
29
4 different fluorescent dyes are attached to 4 separate aliquots of the sample
Dye Primer Sequencing
30
Dye molecules are attached to the 5’ end of the primer = 4 versions of the same primer w/ different dye labels
Dye Primer Sequencing
31
Products are labeled at the 5’ end using the dye color associated w/ the ddNTP at the end of the fragment
Dye Primer Sequencing
32
1 of the 4 fluorescent dyes attached to each of the ddNTPs
Dye Terminator Sequencing
33
All 4 sequencing reactions are performed in the same tube
Dye Terminator Sequencing
34
Product fragments are labeled at the 3’ end
Dye Terminator Sequencing
35
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
Automated Electrophoresis
36
Determines a DNA sequence without having to make a sequencing ladder
PYROSEQUENCING
37
Relies on the generation of light (luminescence) when nucleotides are added to a growing DNA strand
No gels, fluorescent dyes, ddNTPs
PYROSEQUENCING
38
Reaction mix components PYROSEQUENCING
1. ssDNA template 2. Sequencing prime 3. Sulfurylase 4. Luciferase 5. Substrates: adenosine-5’-phosphosulfate (APS) & luciferin
6. 1 of the 4 dNTPs is added to predetermined order of the reaction
39
A.K.A. methylation-specific sequencing
BISULFITE DNA SEQUENCING
40
Chain termination sequencing designed to detect methylated cytosine nucleotides
2-4 µg of genomic DNA is cut with restriction enzymes to facilitate denaturation
BISULFITE DNA SEQUENCING
41
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
BISULFITE DNA SEQUENCING
42
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
RNA SEQUENCING
43
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
DIRECT RNA SEQUENCING
44
A.K.A. massive parallel sequencing
NEXT-GENERATION SEQUENCING (NGS)
45
Designed to sequence large numbers of templates carrying millions of bases
Powerful computer data assembly systems (bioinformatics, computer software and support) are required
NEXT-GENERATION SEQUENCING (NGS)
46
Require the preparation of a sequencing library (sets of DNA fragments representing the regions to be sequenced)
NEXT-GENERATION SEQUENCING (NGS)
47
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
Gene Panels
48
3 TYPES OF Gene Panels
HOT SPOT PANEL
TARGETED PANEL
VERY LARGE PANEL
49
target regions of specific genes known to affect treatment response, disease state, or clinical condition
“Hot-spot” panels
50
critical genes in particular diseases (hematological-cancer specific, solidtumor specific)
Targeted panels
51
diagnostic, prognostic, discovery purposes
Very large panels (>3000 genes)
52
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
NGS Library
53
synthetic short dsDNA carrying sequences complementary to a single primer pair, which may contain short sequences that will ID the sample (indexing / bar coding)
Adapters
54
The regions to be sequenced are enriched by:
1. Probe hybridization
Probes = biotinylated oligonucleotides complementary to specific gene regions
Targeted Libraries
55
loss of library fragments from the sequenced regions
Allele dropout
56
4 NGS Platforms
1. Ion-conductance
2. Reversible dye terminator sequencing
3. Sequencing by ligation
4. Nanopore sequencing
57
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
Ion-Conductance
58
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
Reversible Dye Terminator Sequencing
59
Uses a pool of labeled oligonucleotide DNA ligase to identify the template sequence through the known probe sequences
Sequencing by Ligation
60
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
Nanopore Sequencing
61
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
Data Analysis
62
based on comparison w/ the reference sequence (SNVs, indels, rearrangement sequences, CNVs)
Variant ID
63
Sequence variations from the reference are arranged in a
variant call file (VCF)
64
performed for critical variants ID
Annotations
65
t or f
Confidence in the variant call is determined by sequence quality & coverage = at least 500x (recommended)
t
66
Involves using computer technology (in silico) to collect, store, analyze, & disseminate biological data & information (computational biology)
BIOINFORMATICS
67
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
BASIC LOCAL ALIGNMENT SEARCH
TOOL (BLAST)
68
Assigned a universal nomenclature for mixed, degenerate, or wobble bases
International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular
Biology (IUB)
69
Assigned a universal nomenclature for mixed, degenerate, or wobble bases
International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular
Biology (IUB)
70
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
THE HUMAN GENOME PROJECT (HGP) primary mission
71
THE HUMAN GENOME PROJECT (HGP) established and headed by??
National Institutes of Health (NIH) headed by James Watson
72
1st complete genome sequence (1984)
Epstein-Barr virus
73
_____ (Institute Genomic Research) completed the:
1st sequence of a free-living organism (Haemophilus influenzae)
Sequence of the smallest free-living organism (Mycoplasma genitalium)
Craig Venter & colleague
74
1st sequence of a free-living organism
Haemophilus influenzae
75
Sequence of the smallest free-living organism
Mycoplasma genitalium
