dna sequencing from extraction to information. the process step 1: dna extraction step 1: dna...

DNA SequencingDNA SequencingFrom Extraction to

Information

The ProcessThe Process

Step 1: DNA ExtractionStep 1: DNA Extraction Genomic DNA extraction from the organism Genomic DNA extraction from the organism

((bacteriabacteria)) Step 2: PCR AmplificationStep 2: PCR Amplification

Amplification of the DNA segment of interest Amplification of the DNA segment of interest ((16S gene16S gene))

Step 3: DNA SequencingStep 3: DNA Sequencing Sequencing of the PCR product (Sequencing of the PCR product (amplifiedamplified 16S 16S

genegene))

Step 1: DNA ExtractionStep 1: DNA Extraction

DNA ExtractionDNA Extraction

CellsCellswith DNAwith DNA

DNADNA

Step 2: PCR Step 2: PCR AmplificationAmplification

DNADNA

Target Target GeneGene


PCRPCRAmplificationAmplification

PCR Product(Amplified

Target Gene)

Step 3: DNA SequencingStep 3: DNA Sequencing

PCR product(Amplified

Target Gene)

DNA SequencingDNA SequencingSequence of Target Gene

AGCTGCTAAGCTTGAGCTGCTAAGCTTGAGCTTGCACAAGCTAGCTTGCACAAGCTTAGCTTGCAAGCTTTAGCTTGCAAGCTTAGCTTGCAAGCTTGAGCTTGCAAGCTTGCAAGCTTGCAAGCTCAAGCTTGCAAGCTTGCAAGCTTGCAAGTGCAAGCTTGCAAGCTTGCAACGTTGCACTTGCAACGTTGCAAGCTTGCAAGCTTGAGCTTGCAAGCTTGAAGCTTGCAAGCTAAAGCTTGCAAGCTA

Chapter 1:Chapter 1: DNA ExtractionDNA Extraction

The Cell and its The Cell and its ComponentsComponents

30% chemical

s

70% H2O

phospholipids (2%)polysaccharides (2%)

Ions, small molecules (4%)

RNA (6%)

proteins (15%)

DNA (1%)

Three basic steps of DNA Three basic steps of DNA extractionextraction

Disruption Disruption of cell and lysisof cell and lysis Removal Removal of proteins and other of proteins and other

biochemicalsbiochemicals Recovery Recovery of DNAof DNA

Disruption of cell and lysisDisruption of cell and lysis

Cells are broken down into components using a Cells are broken down into components using a Lysis Lysis BufferBuffer containing: containing: EDTAEDTA: disrupts cell membrane and inhibits DNases: disrupts cell membrane and inhibits DNases SDSSDS: denatures proteins and solubilizes cell : denatures proteins and solubilizes cell

membranesmembranes Proteinase KProteinase K: breaks down proteins: breaks down proteins RNase ARNase A: breaks down RNA: breaks down RNA

Solution is incubated at 55ºC 1-3 hours (or overnight)Solution is incubated at 55ºC 1-3 hours (or overnight)


EDTA

SDSProteinase KRNase A EDTA

DNADNA

+ + + +proteinsRNA lipids ions


After lysis, After lysis, cell extractcell extract contains DNA, contains DNA, proteins, and other proteins, and other chemicals/biochemicalschemicals/biochemicals

cell extract

Solid phase binding (silica membrane)Solid phase binding (silica membrane) Cell extract is applied to a silica membrane Cell extract is applied to a silica membrane

columncolumn DNA binds to membraneDNA binds to membrane

all other molecules flow through and are removedall other molecules flow through and are removed

Removal of proteins and Removal of proteins and biochemicalsbiochemicals

cell extractcell extract

Silica membrane

flow-thru

centrifugation

DNADNA bound to membrane

Recovery of DNARecovery of DNA ElutionElution of silica membrane: of silica membrane:

a low-salt buffer or water is added to the a low-salt buffer or water is added to the membranemembrane

bound DNA falls off of the membrane bound DNA falls off of the membrane

elution

DNADNAin solution

centrifugation

DNA bound to membrane

add water (or buffer)

ReviewReview Step 1Step 1: DNA : DNA ExtractionExtraction



DNADNA

Chapter 2:Chapter 2: PCR PCR

AmplificationAmplification

Step 2: PCR Step 2: PCR AmplificationAmplification

DNADNA





Target Gene)

Part IPart I: DNA : DNA PolymerizationPolymerization

DNA Building BlockDNA Building Block

deoxyribose nucleotide triphosphate (dNTP)deoxyribose nucleotide triphosphate (dNTP)

P

sugarsugar

OO

OHOH HH

HHHH

OCH2basebaseP P

5’5’

4’4’

3’3’ 2’2’

1’1’phoshpate groupsphoshpate groups

OOHH

DNA Polymerization: BasicsDNA Polymerization: Basics

GGOOOOP

AA

CCOOOOP

TTOOPOO

OO

TTOOPPP

HH

OO

AAOOPPP

HH

OO

CCOOPPP

HH

Phosphodiester bondPhosphodiester bond

Existing DNA StrandExisting DNA Strand

dNTP

DNA PolymerizationDNA Polymerization The synthesis of DNA requires: The synthesis of DNA requires:

DNA templateDNA template PrimerPrimer: short oligonucleotide : short oligonucleotide

necessary for DNA polymerase to necessary for DNA polymerase to start start

DNA polymeraseDNA polymerase: enzyme that : enzyme that constructs the DNA chainconstructs the DNA chain

deoxyribonucleotide triphosphatesdeoxyribonucleotide triphosphates ((dNTPsdNTPs): building blocks of DNA): building blocks of DNA

C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C GG A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C

DNA DNA polymerasepolymerase

A C C G G G A A G C C C C G

G A T G A


CTG

A

DNA Replication ReviewDNA Replication Review

Step 1Step 1: : Denaturation: Denaturation: separation of the separation of the two strands of the DNA duplextwo strands of the DNA duplex GyraseGyrase pulls apart the strands creating a “replication pulls apart the strands creating a “replication

bubble”bubble” HelicaseHelicase travels down DNA molecule, breaking the travels down DNA molecule, breaking the

hydrogen bonds that hold the two strands togetherhydrogen bonds that hold the two strands together

gyrasegyrase

gyrasegyrase

G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G CC T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C Ghelicasehelicasehelicasehelicase


Step 2: Step 2: AnnealingAnnealing of primers to the DNA of primers to the DNA template strandtemplate strand PrimasePrimase synthesizes small complementary strands of synthesizes small complementary strands of

RNA (“RNA (“primersprimers”) to the single strands of the DNA ”) to the single strands of the DNA templatetemplate

G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C

C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

G C C C C G

G A T G A G

primaseprimase

primaseprimase



G C C C C G

G A T G A G

Step 3: Step 3: ExtensionExtension of newly constructed of newly constructed complementary DNA moleculescomplementary DNA molecules DNA polymeraseDNA polymerase adds bases to the ends adds bases to the ends

of the primers, constructing an exact copy of the primers, constructing an exact copy of the templateof the template


T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C

C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A





G C C C C G

G A T G A G

Another DNA Polymerase replaces the Another DNA Polymerase replaces the RNA primer with dNTPsRNA primer with dNTPs The final result: two copies of replicated The final result: two copies of replicated

DNADNA


T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C


G A T G A GDNA polymerase

DNA polymerase

DNA Polymerase

AA

O

P

TT

OO

P

AA

OO

P

GG

OO

P

CC

OO

P

TT

OO

P

OO

PolymerizationPolymerization

TT

PO

O

CC

PO

O

GG

PO

O

AA

PO

O

AA

PO

O

CC

PO

O

GG

PO

O

TT

PO

O

AA

PO

O

TT

PO

O

CC

PO

O

GG

PO

O

AA

PO

O

PO

O

PP

CC

O

PO

Mg2+

PP

GG

O

PO

Mg2+

PP

TT

O

PO

Mg2+

PP

TT

O

PO

Mg2+

PP

CC

O

PO

Mg2+

PP

GG

O

PO

Mg2+

PP

AA

O

PO

Mg2+

DNA TemplateDNA Template

1)1) DNA TemplateDNA Template

PrimerPrimer

2)2) PrimerPrimer

DNA PolymeraseDNA Polymerase

3)3) DNA PolymeraseDNA Polymerase4)4) dNTPsdNTPs5)5) MgMg2+ 2+ ionsions

dNTPsdNTPsMgMg2+2+

ionsions

Phosphodiester bondPhosphodiester bond

Part II: PCRPart II: PCR

The Polymerase Chain The Polymerase Chain ReactionReaction

Polymerase Chain Reaction: Polymerase Chain Reaction: cycling process cycling process consisting of the same 3 steps of DNA consisting of the same 3 steps of DNA replication, with some differences:replication, with some differences: temperature cyclingtemperature cycling removes the need for other removes the need for other

enzymes (gyrase/helicase, or primase)enzymes (gyrase/helicase, or primase) PCR uses pre-made oligonucleotide PCR uses pre-made oligonucleotide DNA primersDNA primers

gyrasegyrase

helicasehelicase

primaseprimaseDNA DNA

polymerasepolymeraseT C A G T A

T A G T A C


DuringDuring PCR, PCR, a thermocycler brings the reaction a thermocycler brings the reaction mix to 3 different temperatures analagous to the mix to 3 different temperatures analagous to the 3 steps of DNA replication3 steps of DNA replication DenaturationDenaturation (94˚C) of the DNA template by heat (94˚C) of the DNA template by heat AnnealingAnnealing (37˚-70˚C) of the primers to the template (37˚-70˚C) of the primers to the template ExtensionExtension (72˚C) of the DNA strand by DNA polymerase (72˚C) of the DNA strand by DNA polymerase

These steps are repeated for 25 to 30 cyclesThese steps are repeated for 25 to 30 cycles

94˚C94˚C

denaturationdenaturation

65˚C65˚C

annealingannealing

72˚C72˚C

extensionextension

Thermocycler ProgramThermocycler Program Initial Denaturation: Initial Denaturation: 94˚C94˚C 2 min2 min

Start CycleStart Cycle DenaturationDenaturation 94˚C94˚C 30 sec30 sec AnnealingAnnealing 65˚C65˚C 30 sec30 sec ExtensionExtension 72˚C72˚C 30 sec30 sec

Repeat Cycle 29 times (total = 30 cycles)Repeat Cycle 29 times (total = 30 cycles)

Final ExtensionFinal Extension 72˚C72˚C 7 min7 min HoldHold 4˚C4˚C ∞∞

DenaturationDenaturation

DenaturationDenaturation occurs at 94 occurs at 94˚C˚C The high temperature is used to break down the The high temperature is used to break down the

hydrogen bonds that hold the two strands togetherhydrogen bonds that hold the two strands together

G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G CC T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

94˚C94˚C

AnnealingAnnealing AnnealingAnnealing occurs at 37˚-70˚C occurs at 37˚-70˚C

Oligonuclotide DNA primers anneal to their Oligonuclotide DNA primers anneal to their complementary sequences on the template strandscomplementary sequences on the template strands

Annealing temperature depends on the melting Annealing temperature depends on the melting temperature (Ttemperature (Tmm) of the primer (dependent on base ) of the primer (dependent on base composition)composition)

65˚C65˚CG A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C


T A G T A C

T C A G T A

94˚C94˚C

ExtensionExtension ExtensionExtension occurs at 72˚C occurs at 72˚C

DNA polymerase attaches to the primers and DNA polymerase attaches to the primers and extends the new DNA strandextends the new DNA strand

The 3 steps (The 3 steps (denaturationdenaturation, , annealingannealing, and , and extensionextension) are repeated for another 24 to 29 ) are repeated for another 24 to 29 cyclescycles

72˚C72˚CG A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C


T A G T A C

T C A G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C

C T A C T C A A G C A C A G G C A T G T T G A C C G C A T



65˚C65˚C

Forward PrimerForward Primer

Reverse PrimerReverse Primer

Target SequenceTarget Sequence A desired target sequence is identifiedA desired target sequence is identified To isolate the target sequence, primers To isolate the target sequence, primers

that flank the region must be constructedthat flank the region must be constructed The DNA segment that is then amplified The DNA segment that is then amplified

contains the region of interestcontains the region of interestTemplate DNATemplate DNA

Target Sequence of interestTarget Sequence of interest

PCR ProductPCR Product

PCR: Cycle 1PCR: Cycle 1

Target Sequence of interestTarget Sequence of interest

DNA DNA CopiesCopies

Target Target CopiesCopies

44

00

DenaturationDenaturationAnnealingAnnealingExtensionExtension




88

22





1616

88





3232

2222





6464

5252


PCR Amplification:PCR Amplification: First First 1010 cyclescyclesPCR Amplification

0

200

400

600

800

1,000

1,200

1 2 3 4 5 6 7 8 9 10

Cycle

Copies

DNA copies

T arget Copies

PCR Amplification:PCR Amplification: First First 1515 cyclescycles

PCR Amplification

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Cycle

Copies

DNA copies

T arget Copies

PCR Amplification:PCR Amplification: After After 3030 cyclescycles

PCR Amplification

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Cycle

Copies

DNA copies

Target Copies

Cycle Target Copies Cycle Target Copies

1 0 16 65,504

2 0 17 131,038

3 2 18 262,108

4 8 19 524,250

5 22 20 1,048,536

6 52 21 2,097,110

7 114 22 4,194,260

8 240 23 8,388,562

9 494 24 16,777,168

10 1,004 25 33,554,382

11 2,026 26 67,108,812

12 4,072 27 134,217,674

13 8,166 28 268,435,400

14 16,356 29 536,870,854

15 32,738 30 1,073,741,764

PCR Amplification:PCR Amplification: After 30 After 30 cyclescycles

ReviewReview Step 1Step 1: DNA : DNA ExtractionExtraction



DNADNA

ReviewReview Step 2Step 2: PCR : PCR AmplificationAmplification

DNADNA





Target Gene)

Chapter 3:Chapter 3:DNA SequencingDNA Sequencing

NucleotidesNucleotides

OO

OHOH OHOHHHHH

OCH2

ribose NTP (NTP)ribose NTP (NTP)(Makes up RNA)(Makes up RNA)

BASEBASEPPPOO

OHOH HHHHHH

OCH2

deoxydeoxyribose NTP (ribose NTP (ddNTP)NTP)(Makes up DNA)(Makes up DNA)

BASEBASEPPP

didideoxydeoxyribose NTP (ribose NTP (ddddNTP)NTP)

OO

HH HHHHHH

OCH2BASEBASEPPP

DNA SequencingDNA Sequencing Dideoxy methodDideoxy method of DNA sequencing (Sanger of DNA sequencing (Sanger

Method)Method) Single-stranded DNA to be sequenced serves as a Single-stranded DNA to be sequenced serves as a

template strand for DNA synthesistemplate strand for DNA synthesis single primer is used for DNA synthesis initiationsingle primer is used for DNA synthesis initiation use of dNTPs along with use of dNTPs along with labeled ddNTPslabeled ddNTPs

OO

OHOH HHHHHH

OCH2

dNTPdNTP ddNTPddNTP

BASEBASEPPPOO

HH HHHHHH

OCH2BASEBASEPPP

DNA Polymerization using DNA Polymerization using ddNTPsddNTPs

OO

GGOOOOP

AA

CCOOOOP

TTOOPOO

OO

TTOOPPP

HH

HH

OO

AAOOPPP

HH

OO

CCOOPPP

HH

OO

GGOOOOP

AA

CCOOOOP

CCOOPOO

HH

TTOOPPP

HH

OO

AAOOPPP

HH

Chain TerminationChain Termination

Sequence ReactionSequence Reaction BigDye Terminator v3.1 Sequencing:BigDye Terminator v3.1 Sequencing:

a Dye Terminator Cycle Sequencing a Dye Terminator Cycle Sequencing Master Mix is used for sequencing Master Mix is used for sequencing reaction. reaction.

Components include:Components include: DNA polymerase I, MgDNA polymerase I, Mg2+2+, buffer, buffer dNTPsdNTPs in ample quantities: in ample quantities:

(dATP, dTTP, dCTP, dGTP)(dATP, dTTP, dCTP, dGTP) ddNTPsddNTPs in limited quantities, each labeled in limited quantities, each labeled

with a “tag” that fluoresces a different with a “tag” that fluoresces a different “color”: “color”:

((ddATPddATP, , ddTTPddTTP, , ddCTPddCTP, , ddGTPddGTP))


PCR PCR makes use of a thermocycler to bring the makes use of a thermocycler to bring the reaction mix to three different temperaturesreaction mix to three different temperatures DenaturationDenaturation (94˚C) of the DNA template by heat (94˚C) of the DNA template by heat AnnealingAnnealing (37˚-70˚C) of the primers to the template (37˚-70˚C) of the primers to the template ExtensionExtension (72˚C) of the DNA strand by DNA (72˚C) of the DNA strand by DNA

polymerasepolymerase

These steps are repeated for 25 to 30 These steps are repeated for 25 to 30 cyclescycles

94˚C94˚C

denaturationdenaturation

65˚C65˚C

annealingannealing

72˚C72˚C

extensionextension

Sequencing ReactionSequencing Reaction

Sequencing reactionSequencing reaction is a cycled reaction is a cycled reaction using a thermocycler (as in the using a thermocycler (as in the Polymerase Chain Reaction)Polymerase Chain Reaction)

Like PCR, it consists of 3 steps: Like PCR, it consists of 3 steps: DenaturationDenaturation, , AnnealingAnnealing, , ExtensionExtension; ; these 3 steps are repeated for 30 cyclesthese 3 steps are repeated for 30 cycles

Unlike PCR, it involves a Unlike PCR, it involves a single primersingle primer and and labeled ddNTPslabeled ddNTPs extension proceeds normally until, by chance, extension proceeds normally until, by chance,

DNA polymerase inserts a ddNTP, terminating DNA polymerase inserts a ddNTP, terminating the chainthe chain


C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C GG A A C T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C GG T G T C C G T A C A A C T G G C GG T G T C C G T A C A A C T G G C G T A A T



C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C GG A A C T C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C

G T G T C C G T A C A A C T G G C G T A A T C A T G G

G T G T C C G T A C A A C T G G C G T A A T C A T G

G T G T C C G T A C A A C T G G C G T A A T C A T

G T G T C C G T A C A A C T G G C G T A A T C A

G T G T C C G T A C A A C T G G C G T A A T C

G T G T C C G T A C A A C T G G C G T A A T

G T G T C C G T A C A A C T G G C G T A A

G T G T C C G T A C A A C T G G C G T A

G T G T C C G T A C A A C T G G C G T

G T G T C C G T A C A A C T G G C G


Applied Biosystems 3130xl Applied Biosystems 3130xl Genetic Analyzer Genetic Analyzer

16-channel 16-channel capillary electrophoresiscapillary electrophoresis capable of various genomic analysis capable of various genomic analysis functionsfunctions

Capillary ElectrophoresisCapillary Electrophoresis the sequencing reactions are loaded into the the sequencing reactions are loaded into the

ABI3130xlABI3130xl Samples are taken up by capillaries containing Samples are taken up by capillaries containing

polyacrylamide gel (Performance Optimized Polymer (POP-7)polyacrylamide gel (Performance Optimized Polymer (POP-7) Fragments are separated by length from shortest to longest Fragments are separated by length from shortest to longest

by electrophoresis by electrophoresis

SamplesSamples

Capillary array

Laser

Detector

- +

ElectrophoresisElectrophoresis ElectrophoresisElectrophoresis is a technique used to separate is a technique used to separate

DNA or protein molecules on the basis of size DNA or protein molecules on the basis of size and chargeand charge

Typical method used for analyzing, identifying Typical method used for analyzing, identifying and purifying DNA fragmentsand purifying DNA fragments

Movement in an Electric Movement in an Electric FieldField

The mobility of molecules in the electrical field is The mobility of molecules in the electrical field is also affected by their overall size or molecular also affected by their overall size or molecular weightweight

––

++

Agarose gel

LowerMolecular

weight

HigherMolecular

weight

1500bp1500bp

1000bp1000bp

DNA Gel ElectrophoresisDNA Gel Electrophoresis

100bp100bp

500bp500bp

400bp400bp

300bp300bp

200bp200bp

600bp600bp

700bp700bp

800bp800bp900bp900bp

1100bp1100bp

1200bp1200bp

400bp

1050bp

650bp

280bp

820bp

These “markers” are run These “markers” are run alongside samples,alongside samples,

Helps determine the Helps determine the length of the PCR length of the PCR samplesample

DNA fragments of DNA fragments of the same length will the same length will migrate through the migrate through the gel at the same rategel at the same rate

ElectrophoresisElectrophoresis As the sample travels through the capillariesAs the sample travels through the capillaries

Shorter fragments have less resistance and migrate Shorter fragments have less resistance and migrate faster faster

Longer fragments have more resistance and move Longer fragments have more resistance and move slowerslower

++ --

Fluorescense detectionFluorescense detection As fragments pass through detector window, the As fragments pass through detector window, the

fluorescent “tag” of the ddNTP is excited by a fluorescent “tag” of the ddNTP is excited by a laserlaser The emission of the “tag” is picked up by a detector and The emission of the “tag” is picked up by a detector and

is translated to a colored peak unique to the nucleotideis translated to a colored peak unique to the nucleotide

CTGA


C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C GG A A C T C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C C

G T G T C C G T A C A A C T G G C G T A A T C A T G G C

G T G T C C G T A C A A C T G G C G T A A T C A T G G

G T G T C C G T A C A A C T G G C G T A A T C A T G

G T G T C C G T A C A A C T G G C G T A A T C A T

G T G T C C G T A C A A C T G G C G T A A T C A

G T G T C C G T A C A A C T G G C G T A A T C

G T G T C C G T A C A A C T G G C G T A A T

G T G T C C G T A C A A C T G G C G T A A

G T G T C C G T A C A A C T G G C G T A

G T G T C C G T A C A A C T G G C G T

G T G T C C G T A C A A C T G G C G


Capillary ElectrophoresisCapillary Electrophoresis As the sample travels through the capillariesAs the sample travels through the capillaries

Shorter fragments have less resistance and migrate Shorter fragments have less resistance and migrate faster faster

Longer fragments have more resistance and move Longer fragments have more resistance and move slowerslower

++ --

Detection of fluorescent Detection of fluorescent tagstags

CC GG TT AA AA AA CC AA CC GG GGCC CC CC TT TT CC GGGG GG GG CC

Final DataFinal Data The final data generated is the complete The final data generated is the complete

chromatogram and the text version of the chromatogram and the text version of the DNA sequenceDNA sequence

ACAACTGGCGTGAATCATGGCCCTTCGGGGCCATTGTTTCTCTGTGGAGGAGTGCCATGACGAAAGATGAACTGATTGCCCGTCTCCGCTCGCTGGGTGAACAACTGAACCGTGATGTCAGCCTGACGGGGACGAAAGAAGAACTGGCGCTCCGTGTGGCAGAGCTGAAAGAGGAGCTTGATGACACGGATGAAACTGCCGGTCAGGACACCCCTCTCAGCCGGGAAAATGT

Review Step 1Review Step 1: DNA : DNA ExtractionExtraction



DNADNA

Review Step 2Review Step 2: PCR : PCR AmplificationAmplification

DNADNA





Target Gene)

Review Step 3Review Step 3: DNA : DNA SequencingSequencing

PCR product(Amplified

Target Gene)

DNA SequencingDNA SequencingSequence of Target Gene

AGCTGCTAAGCTTGAGCTGCTAAGCTTGAGCTTGCACAAGCTAGCTTGCACAAGCTTAGCTTGCAAGCTTTAGCTTGCAAGCTTAGCTTGCAAGCTTGAGCTTGCAAGCTTGCAAGCTTGCAAGCTCAAGCTTGCAAGCTTGCAAGCTTGCAAGTGCAAGCTTGCAAGCTTGCAACGTTGCACTTGCAACGTTGCAAGCTTGCAAGCTTGAGCTTGCAAGCTTGAAGCTTGCAAGCTAAAGCTTGCAAGCTA

BWET.PL4.8F SequenceBWET.PL4.8F Sequence

BWET.PL4.8F SequenceBWET.PL4.8F Sequence GCAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACGGCAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACG

ATAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTGGGATAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAACGATGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATAATAGCTTCGGCTCAAAGAGGGGGACCTCTAATACCGCATAATAGCTTCGGCTCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGCTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTATAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTAGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCAGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCAGTGAGGAAGGTGGTGATGTTAATAGCATCATCATTTGACGTTAGCTGAAGGTGGTGATGTTAATAGCATCATCATTTGACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCCGGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCCG

BLASTBLAST

BLASTBLAST: : BBasic asic LLocal ocal AAlignment lignment SSearch earch TToolool www.ncbi.nlm.nih.gov/blastwww.ncbi.nlm.nih.gov/blast

an unknown sequence can be matched up an unknown sequence can be matched up to known sequences published in GenBankto known sequences published in GenBank Lists all sequences producing significant Lists all sequences producing significant

alignmentsalignments Gene identification Gene identification Organism genus/species Organism genus/species % identity alignment/match% identity alignment/match

BLAST Results of BLAST Results of BWET.PL4.8FBWET.PL4.8F

BLAST Results of BLAST Results of BWET.PL4.8FBWET.PL4.8F

SummarySummary of best match: of best match: Vibrio sp. BISLTS1 Vibrio sp. BISLTS1 16S ribosomal 16S ribosomal

RNA geneRNA gene Identities = 435/436 Identities = 435/436 (99%)(99%)

The EndThe End

dna sequencing from extraction to information. the process step 1: dna extraction step 1: dna...

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