DNA Sequencing

Question 1

DNA Sequencing Methods

Answer 1

Direct
Pyrosequencing
Bisulfite
NGS

Question 2

Direct (2)

Answer 2

Manual
Automated

Question 3

Direct - Manual (2)

Answer 3

Chemical (Maxam-Gilbert)
Sanger

Question 4

Direct - Automated (2)

Answer 4

Dye Primer
Dye Terminator

Question 5

NGS (2)

Answer 5

Illumina*
MinION*

Question 6

Most definitive molecular
method to identify genetic
lesions

Answer 6

DNA Sequencing

Question 7

Elucidates the order or
sequence of nucleotides in
a DNA chain

Answer 7

DNA Sequencing

Question 8

Maxam-gilbert

•______ Sequencing
• Requires_________ samples with one end that is______

Answer 8

Chemical double- or single-
stranded DNA

radioactively labeled
(template)

Question 9

• Chemical Sequencing

• Requires double- or single-
stranded DNA samples with one
end that is radioactively labeled
(template)

Answer 9

Maxam-gilbert

Question 10

Maxam-gilbert
1. Templates are aliquoted to____
2. Each tube is treated with a
different chemical
3. Reduction using_____
4. Evaporation of piperidine
5. Drying and resuspension in
_____
6. _____separation
7. Drying of gel and exposure to
____
8. Analysis of data

Answer 10

4 tubes

piperidine

formamide

Polyacrylamide gel

light

Question 11

Madam Gilbert

• For sequencing, the labeled fragment, or template is aliquotted into_____
• Each aliquot is treated with a different chemical with or w/o high salt
• Upon addition of a strong reducing agent (e.g._____), the single-stranded DNA will break at specific nucleotides
• After reactions, piperidine is evaporated, and contents of each tube are dried and resuspended in_____ for gel loading
• The fragments are then separate dby size on a denaturing____
* The denaturing conditions ( formamide, urea, and heat ) PREVENT the single strands of DNA from hydrogen bonding with one another or folding up so that they migrate through the gel STRICTLY ACCORIDNG TO THEIR____.
Migration speed is important because single-base resolution
is required to interpret the sequence properly.
• After electrophoresis
• Gel apparatus is disassembled
• Gel is removed to a sheet of filter paper; then dried on a gel dryer
• Dried gel is exposed to light-sensitive film
• Wet gels can be exposed directly

Answer 11

four tubes

10% piperidine

formamide

polyacrylamide gel

SIZE

Question 12

4 Base Modifiers used in Maxam-Gilbert Sequencing

Answer 12

Dimethylsulphate

Formic acid

Hydrazine

Hydrazine + salt

Question 13

G
Dimethylsulphate

Answer 13

Methylated G at 4C

Question 14

G+ A
Formic acid

Answer 14

Protonates proteins at 5C

Question 15

T+ C
Hydrazine

Answer 15

Splits pyrimidine rings at 8C

Question 16

C
Hydrazine + salt

Answer 16

Splits only C rings at 8 C

Question 17

• Impractical for high-throughput
sequencing of long fragments

• HAZARDOUS REAGENTS!

Answer 17

Maxam-gilbert

Question 18

Madam Gilbert reagent

• - toxic & flammable

Answer 18

Piperidine

Question 19

Toxic and combustible
Irritant!
Flammable

Answer 19

Madam gilbert base modifiers

Question 20

• Detects products using a
radioactive or fluorescent-
labeled nucleotide

Answer 20

Sanger

Question 21

• Modification of the DNA
replication process

Answer 21

Sanger

Question 22

• Dideoxy chain termination

Answer 22

Sanger

Question 23

Products of a Maxam-Gilbert sequencing reaction.
The gel is read from the_____-_____.
The____ of the fragments gives the order of the nucleotides.

The nucleotides are inferred from the lane in which each band appears.

A or T is indicated by bands that appear in the G + A lane or C
+ T lane, respectively, but not in the G lane or the C lane.

Answer 23

bottom to the top

size

Question 24

An alternative detection strategy is to incorporate 32p- or 35S-labeled deoxynucleotides in the nucleotide sequencing reaction mix
A.k.a INTERNAL LABELING

Answer 24

Sanger

Question 25

SANGER 1. 1:1 mixture of____ and _____ into 4 separate reaction tubes with sequencing enzymes and buffer for polymerase activity 2. All _____ and 1/4_____into each tube (different ddNTP in each of the four tubes) a. Ratio of ______is critical for generation of a readable sequence b._________ → polymerization will terminate ***too frequently early along the template*** c._________ → ***infrequent or no termination will occur*** 3._______ is added and reaction runs for 20 minutes

Answer 25

template and primer All dNTPs and ¼ ddNTPs ddNTPs/dNTPs TOO HIGH CONC. TOO LOW CONC. DNA polymerase

Question 26

4. Addition of a stop buffer 20mM (2) a.______ to chelate cations and stop enzyme activity b._____ to denature the products of the synthesis reaction c. Gel loading dyes (2)

Answer 26

EDTA, Formamide loading dye EDTA Formamide bromophenol blue/xylene cyanol)

Question 27

5. Loading into______ a. Products of each of the four sequencing reactions are loaded into adjacent lanes → labeled A, C, G, or T 6._____ the gel and visualize the fragments (expose to x-ray film) a. Fragment patterns are visualized by the signal on the 32p-labeled primer or nucleotide b. All fragments from a given tube will end in the same ddNTP 7. Analysis of sequencing ladder a. Four-lane gel electrophoresis pattern of the products of the four sequencing reactions is called a_____ b. Read to deduce the DNA sequence

Answer 27

denaturing polyacrylamide gel Dry sequencing ladder

Question 28

Sanger 1.) 1:1 mixture of ______into 4 separate reaction tubes with _____ and_____for polymerase activity 2.) All ____ and _____into each tube 3.) DNA polymerase is added and reaction runs for____ 4.) Addition of a ***stop buffer*** (3) 5.) Loading into_____ 6.) Dry the gel and visualize the fragments 7.) Analysis of_____

Answer 28

template:primer ; sequencing enzymes and buffer dNTPs and ¼ ddNTPs ~20 min EDTA, formamide, loading dye denaturing polyacrylamide gel sequencing ladder

Question 29

Larger bands can be compressed at times, ***limiting the length of the sequence*** and is thus ***harder to read*** Improvements to fluorescent dye technology and the introduction of thermal cyclers have led to automation of sequencing

Answer 29

Sanger

Question 30

An improvement to Sanger Sequencing

Answer 30

AUTOMATED Sequencing

Question 31

Uses ***double-stranded templates*** and cycle sequencing that doesn’t require sequential addition of reagents to start and stop the reaction

Answer 31

Automated sequencing

Question 32

Distinct colors (peak wavelengths) of fluorescence emission pertains to the appropriate nucleotide and is the basis of the read instead of the lane

Answer 32

Automated sequencing

Question 33

Madam Gilbert ___%- polyacrylamide gels ran with _____/_____loading dye

Answer 33

6-20% bromophenol blue/xylene cyanol

Question 34

Sanger Label can be in the ____end of the primer or specific nucleotides have incorporated radioisotopes or fluorescent molecules

Answer 34

5'

Question 35

Sanger - lacks the 3' hydroxyl group needed to form a phosphodiester bond with the phosphate group of another nucleotide.

Answer 35

ddNTP

Question 36

Sanger ________ may be added to the sequencing reaction to ***promote equal incorporation of dNTPs for uniform band intensities*** and to increase relative incorporation of ddNTPs

Answer 36

Manganese

Question 37

Sanger ______ and_____may also be added to avoid secondary structures in the synthesized fragments

Answer 37

Deaza-dGP and deoxyinosine triphosphate

Question 38

AUTOMATED SEQUENCING Dye primer • Four different fluorescent dyes are attached to four separate aliquots of the primer • Products are labeled in the____ end with the dye color associated to the ddNTP at the end of the fragments

Answer 38

5’

Question 39

AUTOMATED SEQUENCING DYE terminator • Fluorescent dyes are covalently attached to each_____ instead of the primer • All four sequencing reactions are performed in the____ • Product fragments are labeled at the____ end

Answer 39

ddNTPs same tube 3’

Question 40

AUTOMATED SEQUENCING • Four different fluorescent dyes are attached to four separate aliquots of the primer • Products are labeled in the 5’ end with the dye color associated to the ddNTP at the end of the fragments

Answer 40

Dye primer

Question 41

AUTOMATED SEQUENCING • Fluorescent dyes are covalently attached to each ddNTPs instead of the primer • All four sequencing reactions are performed in the same tube • Product fragments are labeled at the 3’ en

Answer 41

DYE terminator

Question 42

AUTOMATED After the sequencing reaction, ***excess terminators are removed*** Clean with: columns/ beads or by ***ethanol precipitation*** ***Denaturation*** of the fragments

Answer 42

Preparation of Ladder

Question 43

AUTOMATED Ran on the same lane, avoiding lane-to-lane migration variations Migrating fragments pass a laser beam and then a detector Detectors convert fluorescence to an electrical signal (electropherogram)

Answer 43

Electrophoresis

Question 44

AUTOMATED Dye Blobs bright flashes of fluorescence due to failure to clean the ladder Poor starting material leads to poor quality sequence

Answer 44

Sequence interpretation Reading the sequence

Question 45

Most useful for ***short to moderate sequence analysis*** or ***single nucleotide polymorphisms (SNP) and typing*** rather than for generating new sequences

Answer 45

PYROSequencing

Question 46

***Relies on luminescence*** when nucleotides are added to a growing strand of DNA

Answer 46

PYROSequencing

Question 47

Determines a DNA sequence ***without having to make a sequence ladder***

Answer 47

PYROSequencing

Question 48

REACTION MIX: Single-stranded DNA template, sequencing primer, sulfurylase and luciferase, APS, luciferin

Answer 48

PYROSequencing

Question 49

Bisulfite DNA sequencing • Important as____ of DNA affects cell differentiation and is implicated in several diseases such as cancer • Modification of chain termination sequencing to detect ______and is less concerned with generating a new sequence •______ of genomic DNA is cut with restriction enzymes to facilitate denaturation and the fragments are resolved in AGE • Fragments are denatured by____ and exposed to ______for_____ • Cytosines are deaminated = converted to uracil 5-methyl cytosines. = unchanged

Answer 49

methylation methylated nucleotides 2 to 4 ug heat; bisulfite solution (sodium bisulfate, NaOH, and hydroquinone) 16 to 20 hours

Question 50

• Methylation-specific sequencing

Answer 50

Bisulfite DNA sequencing

Question 51

Bisulfite sequencing Unmethylated C = Methylated C =

Answer 51

converted to uracil unchanged

Question 52

Designed to sequence large numbers of templates simultaneously, yielding hundreds of thousands of sequences in just a few hours

Answer 52

Next gen sequencing: Illumina

Question 53

GOALS: ✔ Make sequencing the ***entire genome accessible*** ✔ Make genomic studies a part of routine in research and the clinic

Answer 53

Next gen sequencing: Illumina