Title: Cloning and Sequencing Explorer Series
1(No Transcript)
2Cloning and Sequencing Explorer Series
3 Instructors
- Stan Hitomi
- Coordinator Math Science
- Principal Alamo School
- San Ramon Valley Unified School District
- Danville, CA
- Kirk Brown
- Lead Instructor, Edward Teller Education Center
- Science Chair, Tracy High School
- and Delta College, Tracy, CA
- Bio-Rad Curriculum and Training Specialists
- Sherri Andrews, Ph.D.
- sherri_andrews_at_bio-rad.com
- Leigh Brown, M.A.
- leigh_brown_at_bio-rad.com
4Partnering with Bio-Rad
Bellarmine University Louisville, Kentucky
Prof. Dave Robinson Dr. Joann Lau
Geospiza Inc. Seattle, Wa
5Why TeachCloning and Sequencing Series?
- Students guide the research process and make
decisions about their next steps - Encompasses a myriad of laboratory skills and
techniques commonly used in research - Students generate original data that may lead to
publications in GenBank - Students formulate scientific explanations using
data, logic, and evidence - Students understand research is a process rather
than a single experiment giving students a
real-life research experience with both its
successes and challenges
6Appropriate courses
- Molecular Biology
- Recombinant DNA Techniques
- Biotechnology
- Molecular Evolution
- Bioinformatics
- Advanced Cell Biology
- Advanced Genetics
- Advanced Plant Biology
- Independent Research
7Laboratory Overview
8Student use the following techniques
- Micropipetting
- DNA extraction
- Gel electrophoresis interpretation
- Polymerase chain reaction
- DNA purification
- Restriction enzyme digests
- Microbiological sterile technique
- Preparing competent bacteria
- DNA ligation
- Heat-shock transformation
- Plasmid DNA isolation
- Sequence analysis
- BLAST searching
- GenBank submission
9Students as Authors of Sequence Data
- Output of the lab is a tangible product
- Publication in Genbank database
-
10DNA Preparation and PCR Amplification of GAPDH
- Students choose plant tissue
- Two rounds of PCR
- Round 1 Use of degenerate primers
- Round 2 Nested PCR
11Benefits of using plants
- Large number of species
- Lots of diversity
- Phylogenetic approaches
- Avoid ethical concerns associated with animals
- No pre-approval
12What is a Housekeeping Gene?
- Highly conserved genes that must be continually
expressed in all tissues of organisms to maintain
essential cellular functions. - Examples
- GAPDH
- Cytochrome C
- ATPase
- ß-actin
13Why use GAPDH?
Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)
- Enzyme of glycolysis
- Structure and reaction mechanism well-studied
- Multitude of sequences
- Highly conserved
14Gene Families
15DNA Extraction
- Use young, fresh plant-tissue
- DNA extraction at room temperature
- Time requirement 30 minutes
- Does not require DNA quantification
16PCR Reactions Initial Nested
- Color-coded PCR primers
- (hallmark of Bio-Rad PCR kits)
- Two positive controls
- Arabidopsis
- pGAP (plasmid DNA)
- One negative control
17Initial PCR is done with degenerate primers
Degenerate primers are a mix of primers with
variable sequences designed to recognize the
GAPDH genes of different plant species
- Use of degenerate primers in the initial PCR
reaction may also result in some non-specific
amplifications
18Nested PCR amplifies only regions within the
GAPDH gene Nested PCR is more specific
19Using Nested PCR to increase your final PCR
product
Initial PCR
Nested PCR
DNA template Genomic DNA
DNA template Initial PCR products
- There is more PCR product from the nested PCR
reactions since there is more specific template
DNA to start from - Results intense, bold band on agarose gel
20PCR results1 agarose gel loaded with 20 µl
initial PCR samples and 5 µl nested PCR samples.
1
2
3
4
5
6
7
8
9
Arabidopsis
Green bean
Lambs ear
pGAP
MW
I
N
I
N
I
N
I
N
2000 bp-
1500 bp-
1000 bp-
500 bp-
21Ligation, Transformation and Plasmid Minipreps
- Choose best PCR products for ligation
- Transformation and selection
- Plasmid Miniprep preparation
22Ligation and Transformation
- Column purification (10 minutes)
- Blunt-end PCR product ligate to pJet vector
(30 minutes) - Preparation of competent bacteria cells (30
minutes) - Efficient heat-shock transformation
- (15 minutes)
23Picking colonies for plasmid minipreps
Each colony is a clonal growth (clones) from one
transformed bacteria
24Plasmid minipreps
- Isolate plasmid DNA (40 minutes)
- Restriction digest (1 hour)
- Electrophorese to confirm inserts
- (20 minutes)
25Analysis of plasmid digests
Bgl II Digest
GAPDH inserts
2.5 kb gt 2.0 kb gt 1.5 kb gt 1.0 kb gt 0.5
kb gt
pJet vector
26Example of a miniprep digestion with Bgl II
pJet vector
GAPDH inserts
Digested and undigested DNA were electrophoresed
on a 1 TAE agarose gel
- Lane 1 500 bp molecular weight ruler
- Lanes 2, 4, 6, 8 minipreps digested with
BglII - Lanes 3, 5, 7, 9 undigested minipreps
-
- Different sizes of inserts suggests different
GAPDH genes were cloned in this ligation - Inserts can vary from 0.52.5 kb depending on
plant species
27Setting up Sequencing Reactions
- Add sequencing primers to DNA
- (10 minutes)
- Load 96-well plate
- Send sealed plate off to sequencing facility for
sequencing
28Setting up Sequencing Reactions
GAPDH gene of interest
Always need to sequence reverse, complementary
strand
pJet cloning vector
29Why use multiple sequencing primers?
- Typical sequencing reactions yield 500-600
bases of sequence. - If the GAPDH insert is longer a single set of
sequencing primers will not lead to the full
sequence.
30Sequencing
- Sanger method of sequencing
- 4 fluorescent dyes- 1 for each base
- DNA fragments separated by CE
- Fragments separated in sequential order
- iFinch screens out low quality sequence
31Bioinformatics
- Two month subscription to genetic analysis
software from Geospiza - Data is stored on iFinch server
- Data can be accessed 24/7
32iFinch Landing Page
33What does iFinch do?
- Upload and store DNA sequence data
- Examine the quality of the sequences
- Screens for GAPDH vector sequences
34iFinch Data Folders
Folder for each student group
sequences per folder (4)
35FinchTV Free application for viewing and
editing chromatograms
http//www.geospiza.com/finchtv.html
36FinchTV Sequence Chromatograms
37Data Analysis
Contiguous sequence
- Need to examine all 4 sequences (2 forward, 2
reverse) - Determine overlap and align sequences
- Run the CAP3 program to assemble the sequence
fragments to a full-length contig or contiguous
sequence
38Assembling the full-length contig
- Students compare 3 sequences
- What is the accurate sequence?
- Usually requires going back to chromatograms
Contiguous sequence
39Example of an alignment
Compare between groups before publishing data
40BLAST Searches
Basic Local Alignment Search Tool, or BLAST
Searches a DNA/protein database for published
sequences that are similar to your sequence
41Additional Analysis
- Assemble full-length contig
- Perform BLAST searches
- Identify and remove intronic regions
- Six-frame translation
- Align multiple contigs from the same species
- Preparing sequence for publication
42Student Authors Great for a resume!
43http//classroom1.bio-rad.ifinch.com/Finch/
Try iFinch
Username BR_guest Password guest
Tutorial movies available
http//www.geospiza.com/ifinchBioRad.html
44Watch the Webinar Playback
Planning Guide available for download
45Webinars
- Enzyme Kinetics A Biofuels Case Study
- Real-Time PCR What You Need To Know and Why You
Should Teach It! - Proteins Where DNA Takes on Form and Function
- From plants to sequence a six week college
biology lab course - From singleplex to multiplex making the most out
of your realtime experiments - explorer.bio-rad.com?Support?Webinars