1

2

Cloning and Sequencing Explorer Series

3

Why Teach

Cloning 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

4

Appropriate courses

• Molecular Biology

• Recombinant DNA Techniques

• Biotechnology

• Molecular Evolution

• Bioinformatics

• Advanced Cell Biology

• Advanced Genetics

• Advanced Plant Biology

• Independent Research

5

Laboratory Overview

Microbial CulturingAntibiotic SelectionSterile Technique

Genomic DNA ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

CloningDirect PCR cloningTransformationLigation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

SequencingAutomated sequencing

BioinformaticsSequence Data EditingContig AssemblyIntron-Exon Prediction

6

Student 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

7

Day 1

Day 2

8

DNA Preparation and PCR Amplification of GAPDH

• Students choose plant tissue

• Two rounds of PCR – Round 1: Use of degenerate primers– Round 2: Nested PCR

Genomic DNA ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

9

Optional: Dependent on time constraints, analyze samples prior to the next steps. This may include agarose gel electrophoresis, fluorometry, or spectrometry.

Optional: If time permits proceed directly to Step 1–2 and set up PCR reactions using freshly extracted genomic DNA.

Chapter 1

1: Nucleic Acid Extraction

10

DNA Extraction

• Use young, fresh plant-tissue

• DNA extraction at room temperature

• Time requirement ~30 minutes

• Does not require DNA quantification

11

12

Benefits of using plants

• Large number of species

• Lots of diversity

• Phylogenetic approaches

• Avoid ethical concerns associated with animals

• No pre-approval

Nucleic Acid ExtractionQuick Guide

13

Chapter 2

14

2: GAPDH PCR

15

Optional: Although this protocol recommends analyzing the PCR products after both rounds of PCR have been completed, PCR results can be assessed using electrophoresis directly after this reaction is complete. Positive controls should yield visible bands. It is possible that some plant genomic DNA will not yield a visible band during the initial round of PCR and yet still be amplified after the second round of nested PCR. Note: If this is done — DO NOT add loading dye directly to the PCR reactions as loading dye may interfere with the subsequent round of PCR.

16

PCR Reactions

Initial

Nested

• Color-coded PCR primers (hallmark of Bio-Rad PCR kits)

• Two positive controls• Arabidopsis• pGAP (plasmid DNA)

• One negative control

17

What 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

18

Why use GAPDH?

Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)

• Enzyme of glycolysis

• Structure and reaction mechanism well-studied

• Multitude of sequences

• Highly conserved

19

Host Cell

Endosymbiotic event

Most algae

gene duplication

Land plants

gene duplication

Mesostigma(small group of green algae)

GAPC

GAPA GAPA/B

GAPC/CPGene Families

20

Initial PCR may result in some amplicons that are non-specific

Because of the degenerate primer used to amplify the GAPDH of various plant species the initial PCR may also result in some non-specific amplifications

21

Nested PCR amplifies only regions within the GAPDH gene

Nested PCR is more specific

PCR Animation

http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html

22

DNA sequence varies between species

Primers need to be designed to account for species variation

• What if you don’t know the exact DNA target sequence?

• How do you design primers?

23

Variation in the Genetic Code

Introns are less conserved since they donot code for protein

Conservative substitution-does not change protein properties

The GAPDH enzyme (protein) is highly conserved but there are variations at the DNA level

24

Degenerate primers are used to account for sequence variation

25

Primers are designed using the consensus sequence Tobacco DQ682459

GATTTCGTTGTGGAATCCACTGGCarrot AY491512 GAGTACATTGTGGAGTCCACTGG

Blue gem X78307 GAGTACGTCGTTGAGTCGACTGG

Tomato AB110609 GACTTCGTTGTTGAATCAACCGG

Snapdragon X59517 GAGTATATTGTGGAGTCCACTGG

Consensus sequence GABTATGTTGTTGARTCTTCWGG

Primer set: GA(GTC)TATGTTGTTGA(GA)TCTTC(AT)GGYield: 12 primers

Reverse primers are designed in the same fashion

Plant Accession SequenceGAPDH Number

26

Degenerate primers have optional bases in specified positions

To increase the probability that the primer will anneal to the target DNA, variable bases are designed into the primer.

27

Multiple oligos comprise the forward primer

GAGTATGTTGTTGA(GA)TCTTC(AT)GGGATTATGTTGTTGA(GA)TCTTC(AT)GGGACTATGTTGTTGA(GA)TCTTC(AT)GG

GA(GTC)TATGTTGTTGAGTCTTC(AT)GGGA(GTC)TATGTTGTTGAATCTTC(AT)GG

GA(GTC)TATGTTGTTGA(GA)TCTTCAGGGA(GTC)TATGTTGTTGA(GA)TCTTCTGG

Position 3 has 3 bases

Position 15 has 2 bases

Position 21 has 2 bases

3 x 2 x 2 = 12

different oligonucleotides comprising the forward primer

28

DNA Isolation and Amplification

To identify differences in GAPDH code we must isolate plant DNA and amplify the gene of interest using PCR first with degenerate primers (primers that account for variation in the DNA code)

A second PCR reaction (Nested PCR) is necessary to amplify the region which contains one of the GAPDH gene sequences (second set of primers are nested inside the initial PCR product sequence)

Biotechnology Explorer PCR primers are color-coded

29

Setting up initial PCR Reactions

30

Setting up Initial PCR Reactions Protocol

1. Add 20 µl of blue mastermix with initial primers to each PCR tube

2. Add 15 µl of sterile water to each tube

3. Add 5 µl of DNA template to the appropriate tube

• Control Arabidopsis gDNA

• Control plasmid DNA

• Test gDNAs

• Negative control

4. Amplify in thermal cycler (Annealing temp 52oC)

31

Results of Initial PCR Reactions

1% agarose gel loaded with (20 µl) initial PCR samples.

Green bean and Lamb’s ear gDNA samples generated using Nucleic Acid Extraction module. Lane 1- 500 bp molecular weight ruler (10 µl),

Lane 2- PCR of control Arabidopsis gDNA with initial primers (20 µl)

Lane 3- PCR of green bean gDNA with initial primers (20 µl)

Lane 4- PCR of lamb’s ear gDNA with initial primers (20 µl)

Lane 5- PCR of pGAP plasmid control with initial primers (20 µl)

Sometimes no amplification is observed with initial PCR with some plants, or much fainter than this gel

500 bp-

1000 bp-1500 bp-2000 bp-

1 2 3 4 5

32

• Use of degenerate primers may not give you an exact match to the target sequence

• Because there are multiple primers in the mix, the primer concentration for the matching primer is lower than normal (1/12th

concentration)

• Problems with initial PCR:– inefficient– non-specific

• Benefits of initial PCR:– cast a wide net– increase the pool of specific products

Why is a Nested PCR reaction necessary?

INITIAL GACTATGTTGTTGAGTCTTCTGGFORWARD PRIMER

Arabidopsis GACTACGTTGTTGAGTCTACTGGGAPC1AT3G04120

33

Exonuclease 1 treatment is needed before the second round of PCR (nested PCR) is done

• The primers that were not incorporated into PCR product in the first reaction must be removed so that they do not amplify target DNA in the second round of PCR.

• Exonuclease I will be added to the PCR products

• The enzyme must be inactivated before proceeding to the nested PCR

34

Exo treatment

Protocol

1. Add 1 µl of exonuclease 1 enzyme to the PCR reactions

2. Incubate 15 min 37°C

3. Incubate 15 min 80°C

4. Dilute 2 µl Exo-treated PCR product in 98 µl water

Note: Thermal cycler can be programmed for exo incubations

35

Nested PCR amplifies only regions within the GAPDH gene

Nested PCR is more specific

36

Setting up Nested PCR Reactions Protocol

1. Mix 20 µl of diluted exo-treated template DNA with 20 µl of yellow mastermix with nested primers

2. For controls, mix 20 µl of control pGAP plasmid and 20 µl of water with 20 µl of yellow mastermix with nested primers

3. Amplify in thermal cycler (Annealing temp 46oC)

37

GAPC

GAP-C2

GAPCP-1

GAPCP-2

Nested GAPDH PCR product

PCR Products

Initial vs. Nested Reactions

38

Using Nested PCR to increase your final PCR product

• 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

DNA template:Genomic DNA

DNA template:Initial PCR products

Initial PCR Nested PCR

Chapter 3

39

3: Electrophoresis

40

Electrophoresis Quick Guide

41

42

PCR results

1% agarose gel loaded with 20 µl initial PCR

samples and 5 µl nested PCR samples.

Arabidopsis Green bean Lamb’s ear pGAPMW

I N I N I N I N

1 2 3 4 5 6 7 8 9

500 bp-

1000 bp-

1500 bp-2000 bp-

43

Laboratory Overview

Microbial CulturingAntibiotic SelectionSterile Technique

Genomic DNA ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

CloningDirect PCR cloningTransformationLigation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

SequencingAutomated sequencing

BioinformaticsSequence Data EditingContig AssemblyIntron-Exon Prediction

44

Day 1

Day 2

45

• Choose best PCR products for ligation

• Transformation and selection

• Plasmid Miniprep preparation

Microbial CulturingAntibiotic SelectionSterile Technique Cloning

Direct PCR cloningTransformationLigation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

PCR Purification,Ligation, Transformation and Plasmid Miniprep Purification

Chapter 4

4: PCR Purification

46

Additional tasks to perform prior to next stage:

Starter cultures must be inoculated one day prior to the transformation with a starter colony from the HB101 LB agar starter plate. Incubate cultures with shaking overnight at 37oC.

47

Optional: Electrophorese 5 µl of the purified sample along with 5 µl of the unpurified sample on an agarose gel.

Column Purification of PCR products

48

SEC is used to purify large PCR products from the smaller primers, dNTPs and enzymes.

PCR Purification Quick Guide

49

50

Ligation and Transformation

• Blunt-end PCR product & ligate to pJet vector

• Preparation of competent bacteria cells • Efficient heat-shock transformation

51

Chapter 5

5: Ligation

52

53

Ligation

Sticky End Ligation:If the insert has sticky ends then the vector should be cut with the same enzyme to produce complementary ends

Allows for directional cloning (cDNA)

Blunt End Ligation:All DNA ends are compatible, not necessary to cut vector and insert with the same restriction enzymes

Treating the PCR product with a proofreading DNA polymerase removes 3’-A added by Taq DNA polymerase in PCR, leaving blunt ends ready for ligation.

54

pJet1.2 Blunted Vector

2974 bp in length

Designed for blunt-end cloning MCS (Multiple Cloning Site) has restriction enzyme sites may be used for later manipulation

High copy number

-lactamase gene for Amp resistance

Contains the eco47IR gene

55

Positive Selection

The eco471R gene codes for the Eco47I restriction enzyme which is toxic to E.coli.

When the eco471R gene is disrupted by the insertion of DNA into the cloning site, the gene will no longer be expressed and the transformed cells will grow on selective media.

56

Possible LigationProducts

Expected Results:

Self-ligation of vector

Ligation of vector with multiple inserts

Ligation of vector with primer-dimers or short DNA fragments

Self-ligation of insert

Ligation of one insert into vector

57

Ligation Quick Guide

58

Chapter 6

6: Transformation

59

Transformation

Competent cells

pGAP plasmid control

IPTG

Heat ShockCompetent Cells are very delicate: keep on ice

Bacterial transformation with ligation mixtures is very inefficient (less efficient than transformation with plasmid DNA). Transform with pGAP plasmid as control

Isopropyl -D-1-thiogalactopyranoside (IPTG) is added to the selective medium to artificially increase the expression of the ampr gene and increase transformation efficiency

Heat Shock: plating bacteria from ice to agar plates at 370C

61

Transformation Quick Guide

62

Chapter 77: Plasmid Purification

63

Plasmid Minipreps

• Isolate plasmid DNA

• Restriction digest

• Electrophorese to confirm inserts

64

65

Picking colonies for plasmid minipreps

Each colony is a clonal growth (clones)from one transformed bacteria

66

Plasmid Purification and Restriction Digest AnalysisQuick Guide

67

Optional: It is recommended that 5 µl of undigested DNA also be run next to your digested samples. Prepare these samples by combining 5 µl of miniprep DNA with 5 µl of sterile water.

68

2.5 kb >2.0 kb >

1.5 kb >

1.0 kb >

0.5 kb >

Analysis of plasmid digests

pJet vector

GAPDH inserts

Bgl II Digest

69

Example of a miniprep digestion with Bgl II

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 0.5–2.5 kb depending on plant species

1 2 3 4 5 6 7 8 9

Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel

pJet vector

GAPDH inserts

70

Chapter 8

8: Sequencing

71

Sequencing• Primers, control plasmid and barcoded 96-well

plate are included. Actual sequencing service is not included with the purchase of the series.

• Bio-Rad Laboratories has been working with Eurofins MWG/Operon which is a company that offers sequencing services around the world. We have been able to partner with Eurofins MWG/Operon so that they can offer a highly discounted rate. The benefit of being able to utilize the discounted services of Eurofins MWG/Operon is that their turnaround time is 1-2 days and as an ISO9001 company they adhere to specific quality standards. To contact Eurofins MWG/Operon please visit www.operon.com/bio-rad or call (800)688-2248.

• Alternate options for sequencing would include local universities and other professional sequencing services.

Sequencing Methods • Maxam-Gilbert: Chemical Degradation

– 5’-end of DNA labeled with a radioactive tag– Labled DNA is divided into 4 test tubes with

chemicals which cleave after a particular base– PAGE to seperate cleaved fragments– X-ray film (autoradiography) – Derive DNA sequence from X-ray film image– Limited to about 100 bases

• Sanger: Chain Termination– Single-stranded template of DNA– Template DNA is divided into 4 test tubes with

• Sequencing primers• DNA polymerase• Labled nucleotides (dNTP’s)• Modified nucleotide, different dideoxynucleotide (ddNTP)

to each reaction tube– Allow for DNA synthesis, when ddNTP is incorporated

polymerisation will stop– PAGE and autoradiography can be used to separate

and visualize fragment sizes and read sequence

74

Setting up Sequencing Reactions

• Add sequencing primers to DNA • Load 96-well plate

• Send sealed plate off to chosen sequencing facility for sequencing

75 pJet cloning vector

Setting up Sequencing Reactions

Always need to sequence reverse, complementary strand

GAPDH gene of interest

76

Why 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.

77

• Automated Sanger method of sequencing

• 4 fluorescent dyes- 1 for each base

• DNA fragments separated by CE

• Fragments separated in sequential order

Sequencing

78

Chapter 9

9: Bioinformatics

79

Bioinformatics

• Two month subscription to genetic analysis software from Geospiza

• Data is stored on iFinch server

• Data can be accessed 24/7