Chapter7 Analyzing DNA Chapter7 Analyzing DNA ＆＆ gene structure, varigene structure, variation ation ＆＆ expressionexpression

§1 sequencing §1 sequencing ＆ ＆ genotyping DNAgenotyping DNA

§2 Identifying genes in cloned DNA §2 Identifying genes in cloned DNA ＆ ＆ establishing their sestablishing their structuretructure

§3 Studying gene expression§3 Studying gene expression

ⅠⅠ.sequencing .sequencing ＆ ＆ genotyping DNAgenotyping DNA

Chemical degradation method for DNA sequenChemical degradation method for DNA sequencing (Maxam cing (Maxam ＆＆ Gillbert,1977)Gillbert,1977)

Enzymatic method for DNA sequencing (Fred SaEnzymatic method for DNA sequencing (Fred Sauger,1977)uger,1977)

◆◆The Enzymatic method, in which the sequence of a siThe Enzymatic method, in which the sequence of a single-stranded DNA molecule is determined by enzymatic ngle-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these synthesis of complementary polynucleotide chains, these

chains terminating at specific nucleotide positionschains terminating at specific nucleotide positions

Enzymatic method for DNA sequencingEnzymatic method for DNA sequencing

(Sanger Method / chain terminator sequencing)(Sanger Method / chain terminator sequencing)

principleprinciple : :

Chain termination DNA sequencing is based on the principlChain termination DNA sequencing is based on the principle that single-stranded DNA molecules that differ in length be that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one anothy just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis .This means that it er by polyacrylamide gel electrophoresis .This means that it is possible to resolve a family of molecules, representing all is possible to resolve a family of molecules, representing all lengths from 10 to 1500 nucleotides, into a series of bands lengths from 10 to 1500 nucleotides, into a series of bands

The polymerase enzyme does not discriminate between dNTPs and ddThe polymerase enzyme does not discriminate between dNTPs and ddNTPs, so the dideoxynucleotide can be incorporated into the growing NTPs, so the dideoxynucleotide can be incorporated into the growing

chain, but it then blocks further elongation because it lacks the 3-hydroxchain, but it then blocks further elongation because it lacks the 3-hydrox

yl group needed to form a connection with the next nucleotideyl group needed to form a connection with the next nucleotide

ddCTP ddCTP why we use ddCTP?why we use ddCTP?

dCTP dCTP

We need the We need the primerprimer when sequencing. when sequencing. whywhy??

the primer is needed because template-dependent DNA the primer is needed because template-dependent DNA polymerases cannot initiate DNA synthesis on a polymerases cannot initiate DNA synthesis on a molecule that is entirely single-stranded: there must be a molecule that is entirely single-stranded: there must be a short double-stranded region to provide a 3 - end onto short double-stranded region to provide a 3 - end onto which the enzyme can add new nucleotides. which the enzyme can add new nucleotides.

Chain termination sequencing requires Chain termination sequencing requires a single-stranded DNA a single-stranded DNA templatetemplateThe template for a chain termination experiment is a The template for a chain termination experiment is a single-stranded version of the DNA molecule to be single-stranded version of the DNA molecule to be sequenced. sequenced.

The DNA can be cloned in a plasmid vectorThe DNA can be cloned in a plasmid vector

The resulting DNA will be double stranded so cannot be The resulting DNA will be double stranded so cannot be used directly in sequencing. Instead, it must be converteused directly in sequencing. Instead, it must be converted into single-stranded DNA by denaturation with alkali or d into single-stranded DNA by denaturation with alkali or by boiling. by boiling.

shortcoming shortcoming :it can be difficult to prepare plasmid DNA th:it can be difficult to prepare plasmid DNA that is not contaminated with small quantities of bacterial Dat is not contaminated with small quantities of bacterial DNA and RNA, which can act as spurious templates or priNA and RNA, which can act as spurious templates or primers in the DNA sequencing experiment. mers in the DNA sequencing experiment.

The DNA can be cloned in a bacteriophage MThe DNA can be cloned in a bacteriophage M13 vector.13 vector.

Obtaining single-stranded DNA by cloning in a bacteriophage M13 vector Obtaining single-stranded DNA by cloning in a bacteriophage M13 vector

polymerasepolymerase Three criterion in particular must be fulfilled by a sequencing enzyme:Three criterion in particular must be fulfilled by a sequencing enzyme:

1.1. High processivity High processivity

must have high processivity so that it does not dissocimust have high processivity so that it does not dissociate from the template before incorporating a chain-terate from the template before incorporating a chain-terminating nucleotide.minating nucleotide.

1.1. Negligible or zero 5 Negligible or zero 5 →→ 3 exonuclease activity 3 exonuclease activity

2.2. Negligible or zero 3 Negligible or zero 3 →→5 exonuclease activity 5 exonuclease activity

※ ※desirable the polymerase does not remove the chain desirable the polymerase does not remove the chain termination nucleotide once it has been incorporated.termination nucleotide once it has been incorporated.

Kelenow enzyme; Taq polymerase; sequenase;Kelenow enzyme; Taq polymerase; sequenase;

two advantages over traditional chain termination two advantages over traditional chain termination sequencingsequencing

1.1. very little template DNA is needed, so the DNA does not have to very little template DNA is needed, so the DNA does not have to be cloned before being sequenced. be cloned before being sequenced.

2.2. uses double-stranded rather than single-stranded DNA as the uses double-stranded rather than single-stranded DNA as the starting material.starting material.

cycle sequencingcycle sequencing

Automated DNA sequencingAutomated DNA sequencing

Automated DNA sequencing using fluorescent primersAutomated DNA sequencing using fluorescent primers

This method uses fluorescence labeling, the DNThis method uses fluorescence labeling, the DNA labeled by incorporating a primer or dNTP whiA labeled by incorporating a primer or dNTP which carries a fluorescence. Unlike conventional Dch carries a fluorescence. Unlike conventional DNA sequencing, this method use of different fluorNA sequencing, this method use of different fluorescence in the 4 base, and all 4 reactions can bescence in the 4 base, and all 4 reactions can be loaded into a single lane. During electrophorese loaded into a single lane. During electrophoresis, a monitor detects and records the fluorescencis, a monitor detects and records the fluorescence signal as the DNA passes through a fixed point e signal as the DNA passes through a fixed point in the gel . The output is in the form of intensity pin the gel . The output is in the form of intensity profiles for each of the differently colored fluorophrofiles for each of the differently colored fluorophores , but the information is simultaneously storeores , but the information is simultaneously stored electronically. This precludes transcription errod electronically. This precludes transcription errors when an interpreted sequence is typed by hanrs when an interpreted sequence is typed by hand into a computer file. d into a computer file.

ⅡⅡ. Indentifying genes is cloned DNA . Indentifying genes is cloned DNA ＆＆ establishing their structureestablishing their structure

Exon trappingExon trapping

a technique for detecting sequences within a cloa technique for detecting sequences within a cloned genomic DNA that are capable of splicing to ned genomic DNA that are capable of splicing to exons within a specialized vector. exons within a specialized vector.

This requires a special type of vector that contains a miniThis requires a special type of vector that contains a minigene consisting of two exons flanking an intron sequencgene consisting of two exons flanking an intron sequence, the first exon being preceded by the sequence signals e, the first exon being preceded by the sequence signals needed to initiate transcription in a eukaryotic cell .To usneeded to initiate transcription in a eukaryotic cell .To use the vector the piece of DNA to be studied is inserted inte the vector the piece of DNA to be studied is inserted into a restriction site located within the vector's intron regioo a restriction site located within the vector's intron region. The vector is then introduced into a suitable eukaryotin. The vector is then introduced into a suitable eukaryotic cell line, where it is transcribed and the RNA produced c cell line, where it is transcribed and the RNA produced from it is spliced. The result is that any exon contained in from it is spliced. The result is that any exon contained in the genomic fragment becomes attached between the upthe genomic fragment becomes attached between the upstream and downstream exons from the minigene. RT-Pstream and downstream exons from the minigene. RT-PCR with primers annealing within the two minigene exonCR with primers annealing within the two minigene exons is now used to amplify a DNA fragment, which is seques is now used to amplify a DNA fragment, which is sequenced. As the minigene sequence is already known, the nnced. As the minigene sequence is already known, the nucleotide positions at which the inserted exon starts and ucleotide positions at which the inserted exon starts and ends can be determined, precisely delineating this exon. ends can be determined, precisely delineating this exon.

cDNA selection/capturecDNA selection/capture

a hybridization-based method for retrieving genomic clones a hybridization-based method for retrieving genomic clones that have counterparts in a cDNA librarythat have counterparts in a cDNA library

principle :principle : cognate cDNAs corresponding to genes found within the Ycognate cDNAs corresponding to genes found within the Y

AC will bind preferentially to the YAC DNA.AC will bind preferentially to the YAC DNA.

*YAC: yeast artificial chromosome*YAC: yeast artificial chromosome

early approaches used immobilized YACs and NOearly approaches used immobilized YACs and NO

W used W used solution hybridization reactionsolution hybridization reaction and and biotin- streptavidin capture methods biotin- streptavidin capture methods

cDNA cDNA selection/captureselection/capture (magnetic bead ) (magnetic bead )

RACE-PCRRACE-PCR(rapid amplification of cDNA ends-(rapid amplification of cDNA ends-

PCR)PCR)

A PCR-based technique for mapping the A PCR-based technique for mapping the end of an RNA molecule.end of an RNA molecule.

5`RACE-PCR5`RACE-PCR

In the simplest form of this method one of the primers is In the simplest form of this method one of the primers is

specific for an internal region close to the beginning of thspecific for an internal region close to the beginning of the gene being studied. This primer attaches to the mRNA e gene being studied. This primer attaches to the mRNA for the gene and directs the first reverse-transcriptase-cafor the gene and directs the first reverse-transcriptase-catalyzed stage of the process, during which a cDNA corretalyzed stage of the process, during which a cDNA corresponding to the start of the mRNA is made . Because onlsponding to the start of the mRNA is made . Because only a small segment of the mRNA is being copied, the expy a small segment of the mRNA is being copied, the expectation is that the cDNA synthesis will not terminate preectation is that the cDNA synthesis will not terminate prematurely, so one end of the cDNA will correspond exactlmaturely, so one end of the cDNA will correspond exactly with the start of the mRNA. Once the cDNA has been y with the start of the mRNA. Once the cDNA has been made, a short poly(A) tail is attached to its 3 - end. The smade, a short poly(A) tail is attached to its 3 - end. The second primer anneals to this poly(A) sequence and, duriecond primer anneals to this poly(A) sequence and, during the first round of the normal PCR, converts the single-ng the first round of the normal PCR, converts the single-stranded cDNA into a double-stranded molecule, which istranded cDNA into a double-stranded molecule, which is subsequently amplified as the PCR proceeds. s subsequently amplified as the PCR proceeds.

Mapping transcription start and sites and defMapping transcription start and sites and defining exon-intron boundariesining exon-intron boundaries

Nuclease S1 protection and primer extension Nuclease S1 protection and primer extension assayassay

Primer extension assayPrimer extension assay

Primer extension is used to map the 5' ends of Primer extension is used to map the 5' ends of DNA or RNA fragments. It is done by annealing DNA or RNA fragments. It is done by annealing a specific oligonucleotide primer to a position doa specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, uwnstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended wisually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either ath reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment thn RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. Dat ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA tempNA polymerase can also be used with DNA templates. lates.

Nuclease S1 protectionNuclease S1 protection

The 5`- or 3`-end of a transcript can be ideThe 5`- or 3`-end of a transcript can be identified by hybridization a longer, end-labelntified by hybridization a longer, end-labeled antisense fragment to the RNA. The hyed antisense fragment to the RNA. The hybrid is treated with nuclease S1 to remove brid is treated with nuclease S1 to remove single-stranded regions, and the remaining single-stranded regions, and the remaining fragment`s size is measured on a gel. fragment`s size is measured on a gel.

ⅢⅢ.Studying gene expression.Studying gene expression

Gene expression screeningGene expression screening

↓↓

TargetTarget

↙ ↘ ↙ ↘

RNA transcripts proteinRNA transcripts protein

↓ ↓ ↓ ↓

Hybridization antibodyHybridization antibody

gene-based expression analyses:gene-based expression analyses:

hybridization analyseshybridization analyses

1.Northern blot hybridization1.Northern blot hybridization

2.Tissue 2.Tissue in situin situ hybridization hybridization

3.Whole mount 3.Whole mount in situin situ hybridization hybridization

PCR analysesPCR analyses

1.RT-PCR1.RT-PCR

2.m RNA differential display2.m RNA differential display

ⅡⅡ.protein-based expression analyses:.protein-based expression analyses:

Immunoblotting (Western blotting) Immunoblotting (Western blotting)

ImmunocytochemistryImmunocytochemistry

→ →immunohistochemistryimmunohistochemistry Immunofluorescence microscopyImmunofluorescence microscopy

Ultrastructural studiesUltrastructural studies

Northern blot hybridizationNorthern blot hybridization

This approach affords low resolution expreThis approach affords low resolution expression patterns by hybridizing a gene or cDssion patterns by hybridizing a gene or cDNA probe to total RNA or poly (A)+ RNA eNA probe to total RNA or poly (A)+ RNA extracts prepared from different tissues or cxtracts prepared from different tissues or cell lines. Because the RNA is size-fractionell lines. Because the RNA is size-fractionated on a gel, it is possible to estimate the ated on a gel, it is possible to estimate the size of transcripts. The presence of multiplsize of transcripts. The presence of multiple hybridization bands in one lane may indie hybridization bands in one lane may indicate the presence of differently sized isofocate the presence of differently sized isoforms.rms.

Tissue in situ hybridizationTissue in situ hybridization In situ hybridization (ISH) is a type of hybridization that In situ hybridization (ISH) is a type of hybridization that

uses a labeled complementary DNA or RNA strand to uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or localize a specific DNA or RNA sequence in a portion or section of tissue.section of tissue.

Whole mount in situ hybridizationWhole mount in situ hybridization

an extension of tissue ISH is to study expression in a an extension of tissue ISH is to study expression in a whole embryo.whole embryo.

and whole mount ISH is a popular methods for tracking and whole mount ISH is a popular methods for tracking expression during development in whole embryos from expression during development in whole embryos from vertebrate organisms.vertebrate organisms.

※※fluorescence in situ hybridization (FISfluorescence in situ hybridization (FISH)H)

for single cell expression profilingfor single cell expression profiling FISH is a cytogenetic technique which can be uFISH is a cytogenetic technique which can be u

sed to detect and localize the presence or absensed to detect and localize the presence or absence of specific DNA sequences on chromosomes. ce of specific DNA sequences on chromosomes. It uses fluorescent probes which bind only to thoIt uses fluorescent probes which bind only to those parts of the chromosome with which they shose parts of the chromosome with which they show a high degree of sequence similarity. w a high degree of sequence similarity.

※※large-scale expression screening using large-scale expression screening using microarraysmicroarrays

Chromosome FISH (fluorescence in situ hybridization)Chromosome FISH (fluorescence in situ hybridization)

mRNA differential displaymRNA differential display

a PCR-based technique for comparing the a PCR-based technique for comparing the mRNA species that are expressed in two rmRNA species that are expressed in two related sources of cells to pick out differentielated sources of cells to pick out differentially expressed genes.ally expressed genes.

the method uses a modified oligo(dT) primer which has a the method uses a modified oligo(dT) primer which has a different single nucleotide or dinucleotide at the 3- end cdifferent single nucleotide or dinucleotide at the 3- end causing it to bind to the poly(A) tail of a ausing it to bind to the poly(A) tail of a subsetsubset of mRNAs. of mRNAs. For example, if the oligonucleotide TTTTTTTTTTTCA(T1For example, if the oligonucleotide TTTTTTTTTTTCA(T11CA)1CA) is used as a primer, it will preferentially prime cDN is used as a primer, it will preferentially prime cDNA synthesis from those mRNAs where the dinucleotide TA synthesis from those mRNAs where the dinucleotide TG precedes the poly(A) tail. The second primer which is G precedes the poly(A) tail. The second primer which is used is usually an arbitrary short sequence (often 10 nucused is usually an arbitrary short sequence (often 10 nucleotides long but, because of mismatching, especially at tleotides long but, because of mismatching, especially at the 5 - end, it can bind to many more sites than expected he 5 - end, it can bind to many more sites than expected for a decamer). The resulting amplification patterns are dfor a decamer). The resulting amplification patterns are deliberately designed to produce a complex ladder of baneliberately designed to produce a complex ladder of bands when size-fractionated in a long polyacrylamide gel .ds when size-fractionated in a long polyacrylamide gel .

Antibody labeling and detection systemAntibody labeling and detection system

the primary antibody is used as an intermethe primary antibody is used as an intermediate molecule and is not linked directly to diate molecule and is not linked directly to a labeled group. Once bound to its target, ta labeled group. Once bound to its target, the primary antibody is in turn bound by a she primary antibody is in turn bound by a secondary reagent which is conjugated to a econdary reagent which is conjugated to a reporter which may be a fluorochrome, an reporter which may be a fluorochrome, an enzyme or colloidal gold.enzyme or colloidal gold.

Antibody labeling-detection for tracking Antibody labeling-detection for tracking protein expressionprotein expression

Immunoblotting (Western blotting) Immunoblotting (Western blotting)

a method to detect protein in a given sample of tissue ha method to detect protein in a given sample of tissue homogenate or extract. omogenate or extract.

first uses gel electrophoresis to separate denatured protfirst uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of theins by mass. The proteins are then transferred out of the gel and onto a membrane where they are "probed" usie gel and onto a membrane where they are "probed" using antibodies specific to the protein. As a result, we can ng antibodies specific to the protein. As a result, we can examine the amount of protein in a given sample and coexamine the amount of protein in a given sample and compare levels between several groups. mpare levels between several groups.

Immunohistochemistry (IHC)Immunohistochemistry (IHC)

Immunohistochemistry refers to the prImmunohistochemistry refers to the process of localizing proteins in cells of a ocess of localizing proteins in cells of a tissue section exploiting the principle otissue section exploiting the principle of Abs binding specifically to Ags in biolf Abs binding specifically to Ags in biological tissues. ogical tissues.

Sometimes used to screen RNA expresSometimes used to screen RNA expressionsion

immunocytochemistryimmunocytochemistry

Immunofluorescence microscopyImmunofluorescence microscopy

This method is used when investigating thThis method is used when investigating the e subcellular locationsubcellular location for a protein of intere for a protein of interest. A suitable fluorescent dye, such as fluost. A suitable fluorescent dye, such as fluorescein or rhodamine is coupled to the desrescein or rhodamine is coupled to the desired antibody, enabling the relevant protein ired antibody, enabling the relevant protein to be localized within the cell by fluorescento be localized within the cell by fluorescence microscopyce microscopy

Ultrastructural studiesUltrastructural studies

Higher resolution still of the intracellular loHigher resolution still of the intracellular localization of a gene product or other moleccalization of a gene product or other molecule is possible using electron microscopy. ule is possible using electron microscopy. The antibody is typically labeled with an elThe antibody is typically labeled with an electron-dense particle, such as colloidal golectron-dense particle, such as colloidal gold spheres.d spheres.

Green fluorescent protein (GFP)Green fluorescent protein (GFP)

the GFP gene is frequently used as a repothe GFP gene is frequently used as a reporter gene rter gene

when the GFP gene was cloned and transfwhen the GFP gene was cloned and transfected into target cells in culture, expressioected into target cells in culture, expression of GFP in heterologous cells was also mn of GFP in heterologous cells was also marked by emission of the green fluorescent arked by emission of the green fluorescent light. light.

GFP ribbon diagramGFP ribbon diagram

expression of GFPexpression of GFP

in HeLa Cells in HeLa Cells

Thank you!Thank you!