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)
GAPDH is a housekeeping gene necessary for survival
GAPDH is an enzyme that is crucial for glycolysis to occur

4. Glycolysis

5. 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

6. 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.

7. Gene Cloning

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

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

15. 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

16. 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

17. Primers
Required for both sides of the target sequence (forward & reverse primer)
Length of primer is generally 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

18. 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

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 Plan 1st round nd round (nested)
Initial Denaturation 95oC for 5 minutes oC for 5 minutes
Then 40 Cycles of:
Denaturation oC for 1 minute oC for 1 minute
Annealing oC for 1 minute oC for 1 minute
Extention oC for 2 minutes oC for 2 minutes
Final Extension oC for 6 minutes oC for 6 minutes
Hold oC forever oC forever

21. Gel Electrophoresis

22. 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.

23. 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)

24. 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

25. 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

26. 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.

27. 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

28. 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

29. 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

30. 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)

31. 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

32. 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)

33. 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.

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

35. 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

36. 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

37. 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

38. 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

39. 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

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.
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

42. Automated Sequencing

43. 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