Cloning and Sequencing

Project overview

Background Project will have you cloning the gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) GAPDH is a housekeeping gene necessary for survival GAPDH is an enzyme that is crucial for glycolysis to occur

Glycolysis

GAPDH can be easily isolated in cells Is made up of four subunits that are either identical (homotetramer) or in pairs of slightly different proteins (heterodimer) Has two domains: amino terminal region binds to NAD+ while the carboxy terminal region has the dehydrogenase activity Does two things: Removes H+ from GAP and transfers it to NAD+ Adds second Phosphate to GAP

GAPDH genes Found in the cytosol (glycolysis) and in the chloroplast as part of photosynthesis Isozymes coded for on nuclear DNA GAPC denotes the gene that codes for cytosolic GAPDH and is the gene that we will study. The GAPC protein is a heterodimer.

Gene Cloning

Big picture for this unit Isolate GAPC gene from plants Amplify the GAPC gene by nested PCR Assess the results of the PCR Purify the PCR product containing GAPC Ligate (insert) GAPC gene into plasmid vector Transform bacteria with new plasmid Isolate plasmid from bacteria Confirm plasmid by restriction digests Prepare plasmid DNA to be sequenced by outside facility Analyze sequence of your GAPC gene using bioinformatics

Nucleic Acid Extraction Task is to separate DNA from rest of the cellular components, including membranes, proteins, and enzymes Must also remain in tact after extraction Plant cells also have a cell wall to disrupt Nucleases can digest DNA Acidic contents of organelles can damage DNA Some plants have polyphenols that bind to DNA rendering it useless for experiments

Basic Steps of DNA Extraction Harvest cells from fresh, young plants Grind cells to physically disrupt tissue & cell walls Lyse cells to disrupt membranes Remove cellular debris by centrifugation Digest remaining cellular proteins

Basic Steps of DNA Extraction Purify DNA by ion-exchange chromatography to remove contaminants Concentrate DNA by ethanol precipitation Determine purity and concentration of DNA with UV Spec

Lysis Buffers EDTA to destabilize the membrane and inhibit nucleases Buffers to maintain pH since acids are released by organelles Detergent to dissolve membrane DTT denatures proteins

Polymerase Chain Reaction Rapidly creates multiple copies of a segment of DNA Uses repeated cycles of DNA synthesis in vitro Used in DNA fingerprinting, kinship analysis, genetic testing for mutations, and infectious disease for diagnosis

PCR Round 0 = 1 copy Round 35 = billions of copies

PCR players DNA template – targeted piece of DNA Primers – small segments of DNA that bind complementary upstream and downstream of the target on the template Taq DNA polymerase – isolated from the Thermus aquaticus bacteria found in hotsprings of Yellowstone Park DNA nucleotides in the form of deoxynucleoside triphosphates (dNTPs) Reaction Buffer – maintains pH for enzymes

General PCR Process Denaturation – split apart the two DNA strands by heating them to 95oC for 1 min Annealing – primers bind to target sequence by cooling reaction to 40-60oC for 1 min Extension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 1 min

Primers Required for both sides of the target sequence (forward & reverse primer) Length of primer is generally 18-30 nucleotides G/C content and intra-complementarity are a concern when designing primers Actually not a single primer for each but a mixture of primers (oligoprimers) if the sequence of the target is not known If amino acid sequence of gene product is used then degenerate primers must be used Initial forward primer is GABTATGTTGTTGARTCTTCWGG B=G/T/C R=G/A (purines) W =A/T

Nested PCR Initial PCR primers are degenerate and based on a consensus sequence The chances that the initial primers will bind to sequences other than the target are high A second set of primers designed to be more specific to GAPC is used They are nested within the initial primers and are not degenerate thus much more specific to the GAPC gene

Nested PCR

Our experiment Set-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 Electrophoresis

PCR purification Small impurities can have a negative effect on the ligation of the PCR product to vector DNA Impurities include unincorporated dNTPs, polymerases, primers and small primer-dimers. A PCR Kleen spin column will remove the impurities in less than 4 min.

Gene Cloning Cloning is the production of exact copies of a piece of DNA. It requires ligating (splicing) the PCR product into a cloning vector – often a plasmid DNA The recombinant DNA of the ligation product can now be put into a cell to propagate (replicated)

Plasmids are good vectors: small (2,000 – 10,000 bp) circular, self-replicating high copy number multiple cloning sites (MCS) selectable markers (Amp-resistance) screening (reporter genes, positive select) control mechanisms (lac operon) can handle the size of the insert

pJet1.3 blunted vector Designed for blunt-end cloning High copy number Contains Amp-resistant gene Contains eco47IR gene which allows for positive selection It is 2,974 bp long

Inserts Sticky ends have single strands of nucleotides on ends and are good for directional inserting Blunt ends have no single strands and thus are easier to insert but are non directional.

Ligation T4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ phosphate on another piece. Requires ATP and Mg+2 Insert to vector DNA ratio should be 1:1 Proofing reading DNA polymerase removes dangling 3’A of PCR product

Products of Ligation Self-ligation of vector Ligation of vector to primer-dimers Ligation of multiple inserts Self-ligation of inserts Ligation of one insert into vector

Transformation Once PCR product (insert) has been ligated into a plasmid, the plasmid be introduced into a living bacterial cell to replicate. Two methods of transformation: Electroporation Heat Shock Both methods make cells competent - able to take up plasmids

Transformation Steps Wash away growth media from cells Place cells in ice cold calcium chloride which most likely hardens the cell membrane Add plasmid to cells Move cells to hot environment (usually 42oC) causes membrane pores to open so plasmid can enter Add nutrient media to cells to allow them to recover from stress Plate cells on selective growth plates (Amp and IPTG (increases expression of ampr gene)

Microbial Culturing Pick a colony from the transformed cells to innoculate a liquid culture Liquid culture (broth) must have selective antibiotic (Amp) in it. Choose a single colony from the plate Under favorable conditions, a single bacteria divides every 20 minutes and will multiply into billions in 24 hours

Plasmid Purification To confirm that the engineered cells have been transformed with the correct DNA Different methods Lysozyme Method Alkaline Cell Lysis Method Column Methods (Aurum)

Plasmid preps Spectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/ml Aurum column can process up to 12 OD●ml of bacterial host cells Cells disrupted with a lysis buffer DNA binds to membrane of column, is washed and then eluted with aqueous buffer.

Restriction Digests DNA cut with restriction enzymes Evolved by bacteria to protect against viral DNA infection Endonucleases -cleave within DNA strands Over 3000 known enzymes

Restriction Digests Each enzyme cuts DNA at a specific sequence= restriction site Many of the restriction sites are 4 or 6-base palindrome sequences Enzyme cuts Fragment 2 Fragment 1

Enzyme 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 Digest Restriction Buffer provides optimal conditions: NaCl provides correct ionic strength Tris-HCl provides proper pH Mg+2 is an enzyme co-factor Body temperature (37oC) is optimal Too hot kills enzyme Too cool takes longer digestion time Ody

DNA Sequencing Determining the exact order of the nucleotide sequence in a DNA molecule. Use to take days, now takes hours Have sequences of entire genones for over 700 organisms

Sanger Method Prepare single-stranded DNA template to be sequenced Divide DNA into four test tubes Add primer to each tube to start DNA synthesis Add DNA polymerase Add labeled deoxynucleotides (dNTP) in excess. Labeled with radioactive or fluorescent tags Add a single type of dideoxynucleotides (ddNTPs) to each tube. When incorporated in sythesized strand, synthesis terminates. Allow DNA synthesis to proceed in each tube Run newly synthesized DNA on a polyacrylamide gel

Reading 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 Sequencing Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye. Now reaction can be run in one tube Use capillary electrophoresis rather than the standard polyacrylamide slab gel. When DNA fragment exits gel, the dyes are excited by a laser and emit a light that can be detected . Produces a graph called a chromatogram or electopherogram

Automated Sequencing

Bioinformatics Computerized databases to store, organize, and index the data and for specialized tools to view and analyze biological data Uses include Evolutionary biology Protein modeling Genome mapping Databases are accessible to the public Allow us to record, compare, or identify a DNA sequence