Chapter6- MolecularGeneticTechniques

Twoobjectsofmolecular&genetictechnologies

• Foranalysis• Forgeneration

Moleculargenetictechnologies!

• DNAgelelectrophoresis• Southernblotting• DNAsequencing• Automation• Northernblotting- insituhybridization• cDNAlibrary• Microarray• Nextgenerationsequencing• Beyondtheinvitroworks

Foranalysis

Moleculargenetictechnologies!

• Cloning• Subcloning (foramplification,mutation,fusionandsoon)

– Enzymecutting,Ligation,PCR,Cellculture,DNApurification,Genetransformation,

• Genetransfection(ex.toCellline)• Transgenicanimals• Viralinfectedanimals• GeneeditingwithCRISPR/CAS9

Formanipulation

Nextsemester!

DNAworks

DNAgelelectrophoresis

Onlybysize…

SouthernblottingA technique for DNA detection developed

earlier by Edwin Southern

Whichsequenceisit?

ConceptofPROBE!

DNAiscomplementary!

Question:Issequence‘A’there?

GenomicDNA

Toolongtoanalyze

Restrictionenzyme

Stickyends

Bluntends

Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place.

SouthernblottingA technique for DNA detection developed

earlier by Edwin Southern

Southernblotting

DNAsequencing

DNAsequencing

FrederickSanger,apioneerofsequencing.SangerisoneofthefewscientistswhowasawardedtwoNobelprizes,oneforthe sequencingofproteins,andtheotherforthesequencingofDNA.

DNAsequencing

• ThinktheDNAstructure…

• Youknowthedetailsthenhowtogetthesequenceinformation?

Hint:usingreplicationprocess

Sangermethod

DNAsequencing

• ThinktheDNAstructure…

• Youknowthedetailsthenhowtogetthesequenceinformation?

Hint:usingreplicationprocess

Sangermethod

DNAsequencing

• ThinktheDNAstructure…

• Youknowthedetailsthenhowtogetthesequenceinformation?

Hint:usingreplicationprocess

Sangermethod

Howtostopthereaction?

DNAsequencingddNTP (ddATP,ddGTP,ddCTP,DDTTP)+radioisotope

Sangermethod

DNAsequencingddNTP (ddATP,ddGTP,ddCTP,DDTTP)+something?

• Fluorescentdye

Sangermethod

DNAsequencingddNTP (ddATP,ddGTP,ddCTP,DDTTP)+FLdyes

Automation!!

Sangermethod

Genomesequencing

Genomiclibrary

RNAworks

Northernblotting

• The northernblot isatechniqueusedinmolecularbiologyresearchtostudygeneexpressionbydetectionofRNA(orisolatedmRNA)inasample.

Northernblotting

RNAisolation

RNAqualitycheck

RNAgelelectrophoresis

Membranetransfer

DetectaRNAsequencebyprobe

Northernblotting

Northernblottingtoinsituhybridization

• IfyouwanttoseetheRNAexpressionintissueitself…

• Hint:immunohistochemistry

Insteadofthemembraneyoucanusethetissueitself!

Northernblottingtoinsituhybridization

Northernblottingtoinsituhybridization

Northernblottingtoinsituhybridization

activity, is required for ApLLP gene induction, and it sug-gests that calcium-induced cell signaling may be linkedwith ApLLP gene expression.

ApC/EBP mRNA Induction by High PotassiumTreatment Requires Protein SynthesisIn the present study, we showed that ApLLP overex-pression induced ApC/EBP mRNA. Thus, we investi-gated whether depolarization-induced endogenousApLLP could also induce ApC/EBP mRNA. To addressthis issue, we used an in situ hybridization assay to ex-amine the ApC/EBP mRNA level of cells treated witha high potassium solution. The expression level ofApC/EBP mRNA 15 min after 100 mM high potassiumtreatment for 5 min was increased to 177.7% 6 7.0%in comparison with the nontreated control group (Fig-ures 4C and 4D). In contrast, there were no increasesin the ApC/EBP mRNA levels 30 min (95.98% 6 19.1%,n = 18) and 2 hr (108.7% 6 14.6%, n = 14) after 100 mMhigh potassium treatment (Figure 4D and data notshown). Thus, these data indicate that high potassiumtreatment induces ApC/EBP mRNA transiently.

Next, we examined whether this induction requiresprotein synthesis because ApC/EBP is known as an im-

mediate early gene (IEG), which is expressed by seroto-nin treatment in a translation-independent manner (Al-berini et al., 1994). To inhibit the protein synthesis, weincubated sensory neurons with 10 mM anisomycin innormal media for 1 hr before treatment with 100 mM po-tassium solution in 10 mM anisomycin/normal media. At15 min after anisomycin and high potassium treatment,induction of ApC/EBP mRNA was completely blocked(115.6% 6 8.6%, n = 9) (Figures 4C and 4D). These re-sults indicate that high potassium-elicited ApC/EBP in-duction requires the protein synthesis, suggesting thatApLLP expression is required for the high potassium-elicited ApC/EBP induction.

Injection of Anti-ApLLP Antibody Blocks ApC/EBPInduction Elicited by High Potassium TreatmentTo specifically address the necessity of ApLLP induc-tion for the ApC/EBP induction, we attempted to blockthe functions of ApLLP by using an ApLLP-specific anti-body. We first confirmed the antibody specificity byWestern blot and immunocytochemistry. Western blotanalysis showed that His6-ApLLP was detected only bythe anti-ApLLP antibody but not by preimmune serum(Figure 5A). Furthermore, we also detected a specific

Figure 2. ApLLP Overexpression Induces ApC/EBP mRNA Levels through Its Promoter

(A) The induction of ApC/EBP mRNA in ApLLP-overexpressing sensory neurons. Ectopic ApLLP expression itself increases the ApC/EBP mRNAlevel. Scale bar, 30 mm.(B) Mean pixel intensity representing the ApC/EBP mRNA level. In comparison with the groups that were not treated (n = 11) or treated with a sin-gle 5-HT pulse (n = 10), the ApLLP-overexpressing group (n = 5) showed induction of ApC/EBP mRNA expression (triple asterisk, p < 0.0001,ANOVA and Newman-Keuls multiple comparison test). In comparison with the groups that were not treated or treated with a single 5-HT pulse,the group that was treated with five 5-HT pulses had a greater increase in the intensity of ApC/EBP mRNA expression (double asterisk, p < 0.01,ANOVA and Newman-Keuls multiple comparison test). However, the ApC/EBP mRNA amount was increased to a greater extent by ApLLP over-expression than by treatment with five 5-HT pulses (p < 0.01, ANOVA and Newman-Keuls multiple comparison test).(C) DNA constructs. C/EBP promoter contains 1 kb of ApC/EBP promoter with one TATA sequence and three CRE sites fused to the firefly lu-ciferase gene. The DCRE promoter has three CRE sequences deleted in the ApC/EBP promoter. The Dpro does not have any promoter sequence.(D) Bar graph represents the effect of ApLLP on the ApC/EBP promoter. Normalized luciferase activity in the ApLLP and C/EBP promoter re-porter-injected neurons (n = 9) was increased in comparison with vehicle-injected neurons (n = 8) (double asterisk, p < 0.01, ANOVA and Tukey’smultiple comparison test). However, ApLLP overexpression did not increase the transcriptional activity of the DCRE promoter and D promoterreporter (n = 3 and n = 3, respectively) in comparison to vehicle and DCRE promoter reporter-injected control group (n = 4). Each bar representsthe mean 6 SEM.

Neuron710

248 www.enjournal.org http://dx.doi.org/10.5607/en.2015.24.3.246

Deok-Jin Jang, et al.

Fig. 1. In situ hybridization of ApPDE4 isoforms in Aplysia abdominal ganglia. ApPDE4-positive neurons in Aplysia ganglia. A1, B1: Dorsal (A1) and ventral (B1) surface of abdominal ganglion. A2, B2: Diagrams summarizing the locations of ApPDE4-positive neurons on the dorsal (A2) and ventral (B2) abdominal ganglia. Pl-Ab, pleural-abdominal connection; BagC, bag cell cluster; SN, siphon nerve; GN, gill nerve; PcN, pericardiac nerve; BrN, brachial nerve; Vn, vulvar nerve. C1, D1. Intensity of gray color of circle indicates the signal intensity of ISH-positive neurons. Scale bar 500 μm.

Fig. 2. In situ hybridization of ApPDE4 isoforms in Aplysia cerebral ganglia. A1, B1: Dorsal (A1) and ventral (B1) surface of cerebral ganglia. A2, B2: Diagrams summarizing the localization of ApPDE4-positive neurons on the dorsal (A2) and ventral (B2) cerebral ganglia. MCC, metacerebral neurons; UL, upper lip nerve; AN, antenna nerve; LL, lower lip nerve, CeBu, cerebro-buccal connection; CePd, cerebro-pedal connection; CePl, cerebro-pleural connection. Intensity of gray color of circle indicates the signal intensity of ISH-positive neurons. Scale bar 500 μm.

Insituhybridizationinneuronalganglion(Jang,Kim&Kaang,2016)

Insituhybridizationinculturedcells(KimHF,2006)

cDNAlibrary

Question

• WhichmRNAsareexpressedinaspecifictypeofcell?

1. Cellisolation2. ExtractthemRNA3. Then?

cDNAlibrary

Whatistheimportantstephere?

HowtomaketheRNAtoDNA?Whichkindofprimer?

AcDNAlibrarycontainsrepresentativecopiesofcellularmRNAsequences.

• ComplementaryDNAs(cDNAs)aresynthesizedfromallthemRNAsexpressedinacelltypeandclonedintoplasmidvectorstogenerateacDNAlibrary.

• Steps1and2:RetrovirusreversetranscriptasesynthesizesastrandofDNAcomplementarytoeachmRNAmolecule(cDNA)offanoligo-dTprimerbasepairedtothemRNApolyA tail.

• Steps3–5:ThecDNA-mRNAhybridmoleculesareconvertedintodouble-strandedcDNAmoleculeswithanoligo-dC�oligo-dG double-strandedregionatoneendandanoligo-dT-oligo-dA double-strandedregionattheotherend.

• Step6:MethylationofthecDNAprotectsitfromsubsequentrestrictionenzymecleavage.

• Step7:Shortdouble-strandedlinkerDNAmoleculescontainingaspecificrestrictionenzymerecognitionsiteareligatedtobothendsofthecDNAs usingbacteriophageT4DNAligase.

• Step8a:RestrictionenzymedigestionspecificfortheattachedlinkergeneratescDNAmoleculeswithstickyends.

• Step8b:PlasmidDNAistreatedwiththesamerestrictionenzymetoproduceappropriatepolylinker stickyends.

• Step9:CutplasmidvectorsandthecollectionofcDNAs aremixedata1:1ratioandjoinedcovalentlybyDNAligase.

• TherecombinantDNAplasmidsaretransformedintoE.colicellsforpropagation.

Ifyouwanttoamplifyit…

YoucanusePCRalso!

Thepolymerasechainreaction(PCR)iswidelyusedtoamplifyDNAregionswithknownflankingsequences.

What’renecessary?

PrimerDNApolymerasedNTPBuffer

PCR

https://www.youtube.com/watch?v=iQsu3Kz9NYo

DNAcanbeamplifiedbyPCRforuseincloning

• PCR:• Primersequencesthatareuniquetotargetflankingregionsaresynthesizedtoincluderestrictionenzymerecognitionsequencesnotinthetargetsequence:

• Primer1– containsaBamHIrecognitionsequence

• Primer2– containsaHindIIIrecognitionsequence

• PCRfor20cycles[Note:onlyoneofthetwostrandsisshownforsimplicity.]

• Cloning:• PCR-amplifiedsequencesarecutwithBamHI andHindIII,generatingstickyends.

• Fragmentsareligatedintopolylinker regionofasimilarlycutplasmid.

• RecombinantvectoristransformedintoE.coli.

WhatdoyouwanttodowithcDNAlibrary?

Cell#1 Cell#2

Makequestions…

Whichgenesareexpresseddifferentially?

UsingDNAchip

NEXTGENERATIONSEQUENCING

GenerationofclustersofidenticalDNAfragmentsattachedtoasolidsupport.

• BillionsofdifferentDNAfragmentscanbesequencedsimultaneouslybymethodsbasedonPCR.

• LigateeachendofDNAfragmentstobesequencedtodouble-strandedlinkers.

• Annealtomatchingprimersthatarecovalentlyattachedtoasolidsubstrate.(Reactionsareoptimizedtoproduceasmanyas3× 109discrete,non-overlappingclustersforsequencing.)

• PCRamplifyfor10cyclestoyield~1000identicalcopiesofeachDNAfragmentlocalizedinasmallclusterandattachedatbothendstothesolidsubstrate.

Usingfluorescent-taggeddeoxyribonucleotidetriphosphatesforsequencedetermination.

• Circledcoloreddots:thecolorchangerevealswhichnucleotidewasaddedtotheDNAfragmentineachreactioncycle.

NGS

https://www.youtube.com/watch?v=fCd6B5HRaZ8

Moleculargenetictechnologies!

• Cloning• Subcloning (foramplification,mutation,fusionandsoon)

– Enzymecutting,Ligation,PCR,Cellculture,DNApurification,Genetransformation,

• Genetransfection(ex.toCellline)• Transgenicanimals• Viralinfectedanimals• GeneeditingwithCRISPR/CAS9

Formanipulation

Nextsemester!

Geneticallymodifiedanimals

Readthispaperandsummarizenextgenerationsequencingmethod

Over the past four years, there has been a fundamental shift away from the application of automated Sanger sequencing for genome analysis. Prior to this depar-ture, the automated Sanger method had dominated the industry for almost two decades and led to a number of monumental accomplishments, including the comple-tion of the only finished-grade human genome sequence1. Despite many technical improvements during this era, the limitations of automated Sanger sequencing showed a need for new and improved technologies for sequenc-ing large numbers of human genomes. Recent efforts have been directed towards the development of new methods, leaving Sanger sequencing with fewer reported advances. As such, automated Sanger sequencing is not covered here, and interested readers are directed to previous articles2,3.

The automated Sanger method is considered as a ‘first-generation’ technology, and newer methods are referred to as next-generation sequencing (NGS). These newer technologies constitute various strategies that rely on a combination of template preparation, sequencing and imaging, and genome alignment and assembly methods. The arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and clinical research. In some respects, the potential of NGS is akin to the early days of PCR, with one’s imagination being the primary limitation to its use. The major advance offered by NGS is the ability to produce an enormous volume of data cheaply — in some cases in excess of one billion short reads per instrument run. This feature expands the realm of experimentation beyond just

determining the order of bases. For example, in gene-expression studies microarrays are now being replaced by seq-based methods, which can identify and quantify rare transcripts without prior knowledge of a particular gene and can provide information regarding alternative splicing and sequence variation in identified genes4,5. The ability to sequence the whole genome of many related organisms has allowed large-scale com-parative and evolutionary studies to be performed that were unimaginable just a few years ago. The broadest application of NGS may be the resequencing of human genomes to enhance our understanding of how genetic differences affect health and disease. The variety of NGS features makes it likely that multiple platforms will coexist in the marketplace, with some having clear advantages for particular applications over others.

This Review focuses on commercially available tech-nologies from Roche/454, Illumina/Solexa, Life/APG and Helicos BioSciences, the Polonator instrument and the near-term technology of Pacific Biosciences, who aim to bring their sequencing device to the market in 2010. Nanopore sequencing is not covered, although interested readers are directed to an article by Branton and colleagues6, who describe the advances and remain-ing challenges for this technology. Here, I present a tech-nical review of template preparation, sequencing and imaging, genome alignment and assembly, and current NGS platform performance to provide guidance on how these technologies work and how they may be applied to important biological questions. I highlight the appli-cations of human genome resequencing using targeted and whole-genome approaches, and discuss the progress

*Human Genome Sequencing Center and Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, N1409, Houston, Texas 77030, USA.‡LaserGen, Inc., 8052 El Rio Street, Houston, Texas 77054, USA.e-mail: mmetzker@bcm.edudoi:10.1038/nrg2626Published online 8 December 2009

Automated Sanger sequencingThis process involves a mixture of techniques: bacterial cloning or PCR; template purification; labelling of DNA fragments using the chain termination method with energy transfer, dye-labelled dideoxynucleotides and a DNA polymerase; capillary electrophoresis; and fluorescence detection that provides four-colour plots to reveal the DNA sequence.

Sequencing technologies — the next generationMichael L. Metzker*‡

Abstract | Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

A P P L I C AT I O N S O F N E X T- G E N E R AT I O N S E Q U E N C I N G

REVIEWS

NATURE REVIEWS | GENETICS VOLUME 11 | JANUARY 2010 | 31

Over the past four years, there has been a fundamental shift away from the application of automated Sanger sequencing for genome analysis. Prior to this depar-ture, the automated Sanger method had dominated the industry for almost two decades and led to a number of monumental accomplishments, including the comple-tion of the only finished-grade human genome sequence1. Despite many technical improvements during this era, the limitations of automated Sanger sequencing showed a need for new and improved technologies for sequenc-ing large numbers of human genomes. Recent efforts have been directed towards the development of new methods, leaving Sanger sequencing with fewer reported advances. As such, automated Sanger sequencing is not covered here, and interested readers are directed to previous articles2,3.

The automated Sanger method is considered as a ‘first-generation’ technology, and newer methods are referred to as next-generation sequencing (NGS). These newer technologies constitute various strategies that rely on a combination of template preparation, sequencing and imaging, and genome alignment and assembly methods. The arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and clinical research. In some respects, the potential of NGS is akin to the early days of PCR, with one’s imagination being the primary limitation to its use. The major advance offered by NGS is the ability to produce an enormous volume of data cheaply — in some cases in excess of one billion short reads per instrument run. This feature expands the realm of experimentation beyond just

determining the order of bases. For example, in gene-expression studies microarrays are now being replaced by seq-based methods, which can identify and quantify rare transcripts without prior knowledge of a particular gene and can provide information regarding alternative splicing and sequence variation in identified genes4,5. The ability to sequence the whole genome of many related organisms has allowed large-scale com-parative and evolutionary studies to be performed that were unimaginable just a few years ago. The broadest application of NGS may be the resequencing of human genomes to enhance our understanding of how genetic differences affect health and disease. The variety of NGS features makes it likely that multiple platforms will coexist in the marketplace, with some having clear advantages for particular applications over others.

This Review focuses on commercially available tech-nologies from Roche/454, Illumina/Solexa, Life/APG and Helicos BioSciences, the Polonator instrument and the near-term technology of Pacific Biosciences, who aim to bring their sequencing device to the market in 2010. Nanopore sequencing is not covered, although interested readers are directed to an article by Branton and colleagues6, who describe the advances and remain-ing challenges for this technology. Here, I present a tech-nical review of template preparation, sequencing and imaging, genome alignment and assembly, and current NGS platform performance to provide guidance on how these technologies work and how they may be applied to important biological questions. I highlight the appli-cations of human genome resequencing using targeted and whole-genome approaches, and discuss the progress

*Human Genome Sequencing Center and Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, N1409, Houston, Texas 77030, USA.‡LaserGen, Inc., 8052 El Rio Street, Houston, Texas 77054, USA.e-mail: mmetzker@bcm.edudoi:10.1038/nrg2626Published online 8 December 2009

Automated Sanger sequencingThis process involves a mixture of techniques: bacterial cloning or PCR; template purification; labelling of DNA fragments using the chain termination method with energy transfer, dye-labelled dideoxynucleotides and a DNA polymerase; capillary electrophoresis; and fluorescence detection that provides four-colour plots to reveal the DNA sequence.

Sequencing technologies — the next generationMichael L. Metzker*‡

Abstract | Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

A P P L I C AT I O N S O F N E X T- G E N E R AT I O N S E Q U E N C I N G

REVIEWS

NATURE REVIEWS | GENETICS VOLUME 11 | JANUARY 2010 | 31

1. Summarizethefigure12. Summarizethefigure2withbox13. Summarizethefigure4withbox1

참고 (etc)

InNextsemester…

• Subcloning (foramplification,mutation,fusionandsoon)

– Enzymecutting,Ligation,PCR,Cellculture,DNApurification,Genetransformation,

Formanipulation

Nextsemester!

Ligationofrestrictionfragmentswithcomplementarystickyends.

BasiccomponentsofaplasmidcloningvectorthatcanreplicatewithinanE.colicell.

DNAcloninginaplasmidvectorpermitsamplificationofaDNAfragment.

AyeastgenomiclibrarycanbeconstructedinaplasmidshuttlevectorthatcanreplicateinyeastandinE.coli

6-16Screeningofayeastgenomiclibrarybyfunctionalcomplementationcanidentifyclonescarryingthenormalformofamutantyeastgene.