Cloning and Cloning and SequencingSequencing

Project overviewProject overview

BackgroundBackground

Project will have you cloning the Project will have you cloning the gene that codes for the enzyme gene that codes for the enzyme glyceraldehyde-3-phosphate glyceraldehyde-3-phosphate dehydrogenase (GAPDH)dehydrogenase (GAPDH)

GAPDH is a housekeeping gene GAPDH is a housekeeping gene necessary for survivalnecessary for survival

GAPDH is an enzyme that is crucial GAPDH is an enzyme that is crucial for glycolysis to occurfor glycolysis to occur

GlycolysisGlycolysis

GAPDHGAPDH can be easily isolated in cells can be easily isolated in cells Is made up of four subunits that are either Is made up of four subunits that are either

identical (homotetramer) or in pairs of identical (homotetramer) or in pairs of slightly different proteins (heterodimer)slightly different proteins (heterodimer)

Has two domains: amino terminal region Has two domains: amino terminal region binds to NAD+ while the carboxy terminal binds to NAD+ while the carboxy terminal region has the dehydrogenase activityregion has the dehydrogenase activity

Does two things:Does two things: Removes H+ from GAP and transfersRemoves H+ from GAP and transfers it to NAD+it to NAD+ Adds second Phosphate to GAPAdds second Phosphate to GAP

GAPDH genesGAPDH genes

Found in the cytosol (glycolysis) and Found in the cytosol (glycolysis) and in the chloroplast as part of in the chloroplast as part of photosynthesisphotosynthesis

Isozymes coded for on nuclear DNAIsozymes coded for on nuclear DNA GAPC denotes the gene that codes GAPC denotes the gene that codes

for cytosolic GAPDH and is the gene for cytosolic GAPDH and is the gene that we will study.that we will study.

The GAPC protein is a heterodimer.The GAPC protein is a heterodimer.

Gene CloningGene Cloning

Big picture for this unitBig picture for this unit Isolate GAPC gene from plantsIsolate GAPC gene from plants Amplify the GAPC gene by nested PCRAmplify the GAPC gene by nested PCR Assess the results of the PCRAssess the results of the PCR Purify the PCR product containing GAPCPurify the PCR product containing GAPC Ligate (insert) GAPC gene into plasmid Ligate (insert) GAPC gene into plasmid

vectorvector Transform bacteria with new plasmidTransform bacteria with new plasmid Isolate plasmid from bacteriaIsolate plasmid from bacteria Confirm plasmid by restriction digestsConfirm plasmid by restriction digests Prepare plasmid DNA to be sequenced by Prepare plasmid DNA to be sequenced by

outside facilityoutside facility Analyze sequence of your GAPC gene Analyze sequence of your GAPC gene

using bioinformaticsusing bioinformatics

Nucleic Acid ExtractionNucleic Acid Extraction

Task is to separate DNA from rest of the Task is to separate DNA from rest of the cellular components, including membranes, cellular components, including membranes, proteins, and enzymesproteins, and enzymes

Must also remain in tact after extractionMust also remain in tact after extraction Plant cells also have a cell wall to disruptPlant cells also have a cell wall to disrupt Nucleases can digest DNA Nucleases can digest DNA Acidic contents of organelles can damage Acidic contents of organelles can damage

DNADNA Some plants have polyphenols that bind to Some plants have polyphenols that bind to

DNA rendering it useless for experiments DNA rendering it useless for experiments

Basic Steps of DNA Basic Steps of DNA ExtractionExtraction

Harvest cells from fresh, young plantsHarvest cells from fresh, young plants

Grind cells to physically disrupt tissue & Grind cells to physically disrupt tissue & cell wallscell walls

Lyse cells to disrupt membranesLyse cells to disrupt membranes

Remove cellular debris by centrifugationRemove cellular debris by centrifugation

Digest remaining cellular proteinsDigest remaining cellular proteins

Basic Steps of DNA Basic Steps of DNA ExtractionExtraction

Purify DNA by ion-exchange Purify DNA by ion-exchange chromatography to remove chromatography to remove contaminantscontaminants

Concentrate DNA by ethanol Concentrate DNA by ethanol precipitationprecipitation

Determine purity and concentration of Determine purity and concentration of DNA with UV SpecDNA with UV Spec

Lysis BuffersLysis Buffers

EDTA to destabilize the membrane EDTA to destabilize the membrane and inhibit nucleasesand inhibit nucleases

Buffers to maintain pH since acids Buffers to maintain pH since acids are released by organellesare released by organelles

Detergent to dissolve membraneDetergent to dissolve membrane DTT denatures proteinsDTT denatures proteins

PPolymerase olymerase CChain hain RReactioneaction

Rapidly creates multiple copies of a Rapidly creates multiple copies of a segment of DNAsegment of DNA

Uses repeated cycles of DNA Uses repeated cycles of DNA synthesis synthesis in vitroin vitro

Used in DNA fingerprinting, kinship Used in DNA fingerprinting, kinship analysis, genetic testing for analysis, genetic testing for mutations, and infectious disease for mutations, and infectious disease for diagnosisdiagnosis

PCRPCR

Round 0 = 1 copy

Round 35 = billions of copies

PCR playersPCR players DNA templateDNA template – targeted piece of DNA – targeted piece of DNA PrimersPrimers – small segments of DNA that – small segments of DNA that

bind complementary upstream and bind complementary upstream and downstream of the target on the downstream of the target on the templatetemplate

Taq DNA polymeraseTaq DNA polymerase – isolated from the – isolated from the Thermus aquaticusThermus aquaticus bacteria found in bacteria found in hotsprings of Yellowstone Parkhotsprings of Yellowstone Park

DNA nucleotidesDNA nucleotides in the form of in the form of deoxynucleoside triphosphates (dNTPs)deoxynucleoside triphosphates (dNTPs)

Reaction BufferReaction Buffer – maintains pH for – maintains pH for enzymesenzymes

General PCR ProcessGeneral PCR Process

DenaturationDenaturation – split apart the two DNA – split apart the two DNA strands by heating them to 95strands by heating them to 95ooC for 1 C for 1 minmin

AnnealingAnnealing – primers bind to target – primers bind to target sequence by cooling reaction to 40-60sequence by cooling reaction to 40-60ooC C for 1 minfor 1 min

ExtensionExtension – Taq Polymerase extends the – Taq Polymerase extends the primers and copies each DNA template primers and copies each DNA template strand by heating to 72strand by heating to 72ooC for 1 minC for 1 min

PrimersPrimers Required for both sides of the target Required for both sides of the target

sequence (forward & reverse primer)sequence (forward & reverse primer) Length of primer is generally 18-30 Length of primer is generally 18-30

nucleotidesnucleotides G/C content and intra-complementarity are a G/C content and intra-complementarity are a

concern when designing primersconcern when designing primers Actually not a single primer for each but a Actually not a single primer for each but a

mixture of primers (oligoprimers) if the mixture of primers (oligoprimers) if the sequence of the target is not knownsequence of the target is not known

If amino acid sequence of gene product is If amino acid sequence of gene product is used then degenerate primers must be usedused then degenerate primers must be used

Initial forward primer isInitial forward primer is GAGABBTATGTTGTTGATATGTTGTTGARRTCTTCTCTTCWWGGGG B=G/T/C R=G/A (purines) W =A/TB=G/T/C R=G/A (purines) W =A/T

Nested PCRNested PCR

Initial PCR primers are degenerate Initial PCR primers are degenerate and based on a consensus sequenceand based on a consensus sequence

The chances that the initial primers The chances that the initial primers will bind to sequences other than the will bind to sequences other than the target are hightarget are high

A second set of primers designed to A second set of primers designed to be more specific to GAPC is usedbe more specific to GAPC is used

They are nested within the initial They are nested within the initial primers and are not degenerate thus primers and are not degenerate thus much more specific to the GAPC genemuch more specific to the GAPC gene

Nested PCRNested PCR

Our experimentOur experimentSet-up

Tube 1: negative control (no DNA)

Tube 2: Arabidopsis gDNA

Tube 3: Positive control pGAP plasmid

Tube 4: Your plant DNA

PCR Plan 1st round 2nd round (nested)

Initial Denaturation 95oC for 5 minutes 95oC for 5 minutes

Then 40 Cycles of:

Denaturation 95oC for 1 minute 95oC for 1 minute

Annealing 52oC for 1 minute 46oC for 1 minute

Extention 72oC for 2 minutes 72oC for 2 minutes

Final Extension 72oC for 6 minutes 72oC for 6 minutes

Hold 15oC forever 15oC forever

Gel ElectrophoresisGel Electrophoresis

PCR purificationPCR purification

Small impurities can have a negative Small impurities can have a negative effect on the ligation of the PCR effect on the ligation of the PCR product to vector DNAproduct to vector DNA

Impurities include unincorporated Impurities include unincorporated dNTPs, polymerases, primers and dNTPs, polymerases, primers and small primer-dimers.small primer-dimers.

A PCR Kleen spin column will A PCR Kleen spin column will remove the impurities in less than 4 remove the impurities in less than 4 min.min.

Gene CloningGene Cloning

Cloning is the production of exact Cloning is the production of exact copies of a piece of DNA.copies of a piece of DNA.

It requires ligating (splicing) the It requires ligating (splicing) the PCR product into a cloning vector – PCR product into a cloning vector – often a plasmid DNAoften a plasmid DNA

The recombinant DNA of the ligation The recombinant DNA of the ligation product can now be put into a cell to product can now be put into a cell to propagate (replicated)propagate (replicated)

Plasmids are good Plasmids are good vectors:vectors:

small (2,000 – 10,000 bp) small (2,000 – 10,000 bp) circular, self-replicating circular, self-replicating high copy numberhigh copy number multiple cloning sites (MCS)multiple cloning sites (MCS) selectable markers (Amp-resistance)selectable markers (Amp-resistance) screening (reporter genes, positive screening (reporter genes, positive

select)select) control mechanisms (lac operon)control mechanisms (lac operon) can handle the size of the insertcan handle the size of the insert

pJet1.3 blunted vectorpJet1.3 blunted vector

Designed for blunt-end cloningDesigned for blunt-end cloning High copy numberHigh copy number Contains Amp-resistant geneContains Amp-resistant gene Contains eco47IR gene which allows Contains eco47IR gene which allows

for positive selectionfor positive selection It is 2,974 bp longIt is 2,974 bp long

InsertsInserts

Sticky ends have single strands of Sticky ends have single strands of nucleotides on ends and are good for nucleotides on ends and are good for directional insertingdirectional inserting

Blunt ends have no single Blunt ends have no single

strands and thus are easierstrands and thus are easier

to insert but are non to insert but are non

directional.directional.

LigationLigation

T4 DNA Ligase catalyzes formation of T4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ hydroxy on one piece and the 5’ phosphate on another piece.phosphate on another piece.

Requires ATP and MgRequires ATP and Mg+2+2

Insert to vector DNA ratio should be Insert to vector DNA ratio should be 1:11:1

Proofing reading DNA polymerase Proofing reading DNA polymerase removes dangling 3’A of PCR productremoves dangling 3’A of PCR product

Products of LigationProducts of Ligation

Self-ligation of vectorSelf-ligation of vector Ligation of vector to primer-dimersLigation of vector to primer-dimers Ligation of multiple insertsLigation of multiple inserts Self-ligation of insertsSelf-ligation of inserts Ligation of one insert into vectorLigation of one insert into vector

TransformationTransformation

Once PCR product (insert) has been Once PCR product (insert) has been ligated into a plasmid, the plasmid ligated into a plasmid, the plasmid be introduced into a living bacterial be introduced into a living bacterial cell to replicate.cell to replicate.

Two methods of transformation:Two methods of transformation: ElectroporationElectroporation Heat ShockHeat Shock

Both methods make cells Both methods make cells competent competent -- able to take up plasmids able to take up plasmids

Transformation StepsTransformation Steps

Wash away growth media from cellsWash away growth media from cells Place cells in ice cold calcium chloride which Place cells in ice cold calcium chloride which

most likely hardens the cell membranemost likely hardens the cell membrane Add plasmid to cellsAdd plasmid to cells Move cells to hot environment (usually 42Move cells to hot environment (usually 42ooC) C)

causes membrane pores to open so plasmid causes membrane pores to open so plasmid can entercan enter

Add nutrient media to cells to allow them to Add nutrient media to cells to allow them to recover from stressrecover from stress

Plate cells on selective growth plates (Amp Plate cells on selective growth plates (Amp and IPTG (increases expression of ampand IPTG (increases expression of amprr gene) gene)

Microbial CulturingMicrobial Culturing

Pick a colony from the transformed Pick a colony from the transformed cells to innoculate a liquid culturecells to innoculate a liquid culture

Liquid culture (broth) must have Liquid culture (broth) must have selective antibiotic (Amp) in it.selective antibiotic (Amp) in it.

Choose a single colony from the plateChoose a single colony from the plate Under favorable conditions, a single Under favorable conditions, a single

bacteria divides every 20 minutes and bacteria divides every 20 minutes and will multiply into billions in 24 hourswill multiply into billions in 24 hours

Plasmid PurificationPlasmid Purification

To confirm that the engineered cells To confirm that the engineered cells have been transformed with the have been transformed with the correct DNAcorrect DNA

Different methodsDifferent methods Lysozyme Method Lysozyme Method Alkaline Cell Lysis MethodAlkaline Cell Lysis Method Column Methods (Aurum)Column Methods (Aurum)

Plasmid prepsPlasmid preps

Spectrophotometer determination of Spectrophotometer determination of culture density. Take ODculture density. Take OD600600 of culture of culture (equal to about 8x10(equal to about 8x1088 cells/ml cells/ml

Aurum column can process up to 12 Aurum column can process up to 12 ODOD●●ml of bacterial host cellsml of bacterial host cells

Cells disrupted with a lysis bufferCells disrupted with a lysis buffer DNA binds to membrane of column, is DNA binds to membrane of column, is

washed and then eluted with aqueous washed and then eluted with aqueous buffer.buffer.

Restriction DigestsRestriction Digests

DNA cut with restriction enzymesDNA cut with restriction enzymes Evolved by bacteria to protect Evolved by bacteria to protect

against viral DNA infectionagainst viral DNA infection Endonucleases -cleave within DNA Endonucleases -cleave within DNA

strandsstrands Over 3000 known enzymesOver 3000 known enzymes

Restriction DigestsRestriction Digests

Each enzyme cuts Each enzyme cuts DNA at a specific DNA at a specific sequence= sequence= restriction siterestriction site

Many of the Many of the restriction sites are restriction sites are 4 or 6-base 4 or 6-base palindrome palindrome sequencessequences

Enzyme cuts

Fragment 1 Fragment 2

Enzyme ExamplesEnzyme Examples

EcoRI G-A-A-T-T-C

C-T-T-A-A-G

HindIII A-A-G-C-T-T

T-T-C-G-A-A

BamHI G-G-A-T-C-C

C-C-T-A-G-G

Bgl II A-G-A-T-C-T

T-C-T-A-G-A

Restriction DigestRestriction Digest

Restriction Buffer provides optimal Restriction Buffer provides optimal conditions:conditions: NaCl provides correct ionic strengthNaCl provides correct ionic strength Tris-HCl provides proper pHTris-HCl provides proper pH MgMg+2+2 is an enzyme co-factor is an enzyme co-factor

Body temperature (37Body temperature (37ooC) is optimalC) is optimal Too hot kills enzymeToo hot kills enzyme Too cool takes longer digestion timeToo cool takes longer digestion time

DNA SequencingDNA Sequencing

Determining the exact order of the Determining the exact order of the nucleotide sequence in a DNA nucleotide sequence in a DNA molecule.molecule.

Use to take days, now takes hoursUse to take days, now takes hours Have sequences of entire genones Have sequences of entire genones

for over 700 organismsfor over 700 organisms

Sanger MethodSanger Method Prepare single-stranded DNA template to be Prepare single-stranded DNA template to be

sequencedsequenced Divide DNA into four test tubesDivide DNA into four test tubes Add primer to each tube to start DNA synthesisAdd primer to each tube to start DNA synthesis Add DNA polymeraseAdd DNA polymerase Add labeled deoxynucleotides (dNTP) in excess. Add labeled deoxynucleotides (dNTP) in excess.

Labeled with radioactive or fluorescent tagsLabeled with radioactive or fluorescent tags Add a single type of dideoxynucleotides Add a single type of dideoxynucleotides

(ddNTPs) to each tube. When incorporated in (ddNTPs) to each tube. When incorporated in sythesized strand, synthesis terminates. sythesized strand, synthesis terminates.

Allow DNA synthesis to proceed in each tubeAllow DNA synthesis to proceed in each tube Run newly synthesized DNA on a Run newly synthesized DNA on a

polyacrylamide gelpolyacrylamide gel

Reading the SequenceReading the Sequence•In the tube with the ddTTP, every time it is time to add a T to the new strand, some Ts will be dTTP and some will be ddTTP.

•When the ddTTP is added, then extension stops and you have a DNA fragment of a particular length.

•The T tube will, therefore, have a series of DNA fragments that each terminate with a ddTTP.

•Thus the T tube will show you everywhere there is a T on the gel

•Same thing happens in all tubes

•Read gel from top to bottom looking at all four lanes to get the sequence.

Automated SequencingAutomated Sequencing Dye-terminator sequencing labels each Dye-terminator sequencing labels each

of the ddNTPs with a different color of the ddNTPs with a different color fluorescent dye.fluorescent dye.

Now reaction can be run in one tubeNow reaction can be run in one tube Use capillary electrophoresis rather than Use capillary electrophoresis rather than

the standard polyacrylamide slab gel.the standard polyacrylamide slab gel. When DNA fragment exits gel, the dyes When DNA fragment exits gel, the dyes

are excited by a laser and emit a light are excited by a laser and emit a light that can be detected .that can be detected .

Produces a graph called a chromatogram Produces a graph called a chromatogram or electopherogramor electopherogram

Automated SequencingAutomated Sequencing

BioinformaticsBioinformatics

Computerized databases to store, Computerized databases to store, organize, and index the data and for organize, and index the data and for specialized tools to view and analyze specialized tools to view and analyze biological databiological data

Uses includeUses include Evolutionary biologyEvolutionary biology Protein modelingProtein modeling Genome mappingGenome mapping

Databases are accessible to the public Databases are accessible to the public Allow us to record, compare, or Allow us to record, compare, or

identify a DNA sequenceidentify a DNA sequence