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Cloning and Sequencing. Project overview Background Project will have you cloning the gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase.

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Presentation on theme: "Cloning and Sequencing. Project overview Background Project will have you cloning the gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase."— Presentation transcript:

1 Cloning and Sequencing

2 Project overview

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

4 Glycolysis

5 GAPDH can be easily isolated in cells 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) 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 Has two domains: amino terminal region binds to NAD+ while the carboxy terminal region has the dehydrogenase activity Does two things: Does two things: Removes H+ from GAP and transfers Removes H+ from GAP and transfers it to NAD+ it to NAD+ Adds second Phosphate to GAP Adds second Phosphate to GAP

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

7 Gene Cloning

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

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

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

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

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

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

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

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

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

17 Primers Required for both sides of the target sequence (forward & reverse primer) Required for both sides of the target sequence (forward & reverse primer) Length of primer is generally 18-30 nucleotides Length of primer is generally 18-30 nucleotides G/C content and intra-complementarity are a concern when designing primers 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 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 If amino acid sequence of gene product is used then degenerate primers must be used Initial forward primer is Initial forward primer is GABTATGTTGTTGARTCTTCWGG GABTATGTTGTTGARTCTTCWGG B=G/T/C R=G/A (purines) W =A/T B=G/T/C R=G/A (purines) W =A/T

18 Nested PCR Initial PCR primers are degenerate and based on a consensus sequence 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 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 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 They are nested within the initial primers and are not degenerate thus much more specific to the GAPC gene

19 Nested PCR

20 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 Plan1 st round 2 nd round (nested) Initial Denaturation95 o C for 5 minutes 95 o C for 5 minutes Then 40 Cycles of: Denaturation 95 o C for 1 minute 95 o C for 1 minute Annealing 52 o C for 1 minute 46 o C for 1 minute Extention 72 o C for 2 minutes 72 o C for 2 minutes Final Extension 72 o C for 6 minutes 72 o C for 6 minutes Hold 15 o C forever 15 o C forever

21 Gel Electrophoresis

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

23 Gene Cloning Cloning is the production of exact copies of a piece of DNA. 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 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) The recombinant DNA of the ligation product can now be put into a cell to propagate (replicated)

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

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

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

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

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

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

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

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

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

33 Plasmid preps Spectrophotometer determination of culture density. Take OD 600 of culture (equal to about 8x10 8 cells/ml Spectrophotometer determination of culture density. Take OD 600 of culture (equal to about 8x10 8 cells/ml Aurum column can process up to 12 OD ● ml of bacterial host cells Aurum column can process up to 12 OD ● ml of bacterial host cells Cells disrupted with a lysis buffer Cells disrupted with a lysis buffer DNA binds to membrane of column, is washed and then eluted with aqueous buffer. DNA binds to membrane of column, is washed and then eluted with aqueous buffer.

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

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

36 Enzyme Examples EcoRIG-A-A-T-T-C C-T-T-A-A-G HindIIIA-A-G-C-T-T T-T-C-G-A-A BamHIG-G-A-T-C-C C-C-T-A-G-G Bgl IIA-G-A-T-C-T T-C-T-A-G-A

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

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

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

40 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.

41 Automated Sequencing Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye. Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye. Now reaction can be run in one tube Now reaction can be run in one tube Use capillary electrophoresis rather than the standard polyacrylamide slab gel. 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. 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 Produces a graph called a chromatogram or electopherogram

42 Automated Sequencing

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


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