Short course on DNA barcoding methodsNovember 29, 2011

PCR amplification

Darío LijtmaerMuseo Argentino de Ciencias Naturales “Bernardino Rivadavia”

Organization of the talk

1) Equipment needed for PCR amplification.

2) Overview of PCR protocols.

3) Product verification: agarose gels.

4) Minimizing the risks of contamination.

5) Shipping and storing DNA extracts.

6) Discussion and questions.

VortexPipettes

Equipment: basic for a small-sized facility

Disposables and reagents

- Hundreds or few thousands of barcodes produced per year.

- Tube scale.

IncubatorThermocycler

Autoclave

Equipment: medium-sized and high throughput facilities

- Many thousand barcodes produced per year.

- Plate scale.

IncubatorThermocycler

Autoclave

VortexPipettes Disposables and

reagents

None of the lab protocols/procedures are necessarily different from those used for other mitochondrial markers or other projects.

Overview of amplification protocols

However...

None of the lab protocols/procedures are necessarily different from those used for other mitochondrial markers or other projects.

a) Due to the scale of the project efforts are made to reduce the cost of the molecular steps of the pipeline (e.g. small PCR volumes).

b) Certain requirements are needed to achieve the barcode data standard (e.g. minimum length).

As a consequence innovations and development of new, more efficient protocols/proceedures are frequent in the context of the project.

Overview of amplification protocols

www.barcodeoflife.org

Overview of amplification protocols: CCDB

This protocol can be used with:

• Individual tubes in small-sized facilities.

• 96 well plates in medium-sized or high throughput facilities.

Animals: COI

This protocol can be used with:

• Individual tubes in small-sized facilities.

• 96 well plates in medium-sized or high throughput facilities.

Plants and fungi

www.barcodeoflife.org

Overview of amplification protocols: CCDB

Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and rbcL in plants), 6.25 ml for matK (plants). • Cost-efficient.

Overview of amplification protocols: PCR mix (CCDB)

Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and rbcL in plants), 6.25 ml for matK (plants). • Cost-efficient.

Trehalose is used as part of the PCR mix.• It allows freezing aliquots of the mix (useful for high throughput

facilities).• It estabilizes the reaction.

Overview of amplification protocols: PCR mix (CCDB)

Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and rbcL in plants), 6.25 ml for matK (plants). • Cost-efficient.

Trehalose is used as part of the PCR mix.• It allows freezing aliquots of the mix (useful for high throughput

facilities).• It estabilizes the reaction.

Platinum taq polymerase.• High success and band intensity, less optimization needed.• Hot start .• Stable at room temperature.

Overview of amplification protocols: PCR mix (CCDB)

Overview of amplification protocols: PCR mix (other tips)

BSA can be added to the PCR mix to improve PCR results. This is done at Smithsonian LAB with invertebrate samples and in the African Centre for DNA Barcoding with plant samples.

Overview of amplification protocols: PCR mix (other tips)

BSA can be added to the PCR mix to improve PCR results. This is done at Smithsonian LAB with invertebrate samples and in the African Centre for DNA Barcoding with plant samples.

DMSO can also be added to improve PCR results with difficult samples.

Overview of amplification protocols: primers

Primer choice is a key aspect of PCR success and probably the only aspect of amplification that is taxon-dependent.

Overview of amplification protocols: primers

Primer choice is a key aspect of PCR success and probably the only aspect of amplification that is taxon-dependent.

Ideal situation: universal primers.

Real world: various sets of primers are to be used (and sometimes combined) depending on the taxonomic group. There is also more than one option for each group.

Overview of amplification protocols: primers

Primer choice is a key aspect of PCR success and probably the only aspect of amplification that is taxon-dependent.

Ideal situation: universal primers.

Real world: various sets of primers are to be used (and sometimes combined) depending on the taxonomic group. There is also more than one option for each group.

Overview of amplification protocols: primers

Primer choice is a key aspect of PCR success and probably the only aspect of amplification that is taxon-dependent.

Ideal situation: universal primers.

Real world: various sets of primers are to be used (and sometimes combined) depending on the taxonomic group. There is also more than one option for each group.

We included as part of the complementary materials:

the list of primers that are used at the CCDB with each taxonomic group, the sequence of those primers and the thermocycling program that is used with each primer.

the list of primers and the thermocycling program used at the Smithsonian LAB.

Overview of amplification protocols

PCR protocols also depend on the quality of the samples used.

Overview of amplification protocols

PCR protocols also depend on the quality of the samples used.

For example, samples with potentially degraded DNA, such as relatively old museum samples, require special primers designed to amplify shorter, overlapping fragments (e.g. 200 bp long).

Overview of amplification protocols

PCR protocols also depend on the quality of the samples used.

For example, samples with potentially degraded DNA, such as relatively old museum samples, require special primers designed to amplify shorter, overlapping fragments (e.g. 200 bp long).

Product verification: agarose gels

PCR results are usually visualized in agarose gels.

Depending on the scale (and funding) home made gels or pre-cast gels are used.

PCR results are usually visualized in agarose gels.

Depending on the scale (and funding) home made gels or pre-cast gels are used.

For plate-scales (medium scale or high-throughput) usually a threshold is established (e.g. 75/95 bands at CCDB) and when a plate results are above the threshold the entire plate is sequenced .

Product verification: agarose gels

Mailing PCR products

If sequencing is not done on-site, PCR products are transferred to a plate containing trehalose and dried before mailing them to a high throughput facility.

Minimizing the risk of contamination

General practices

Clean workspace and sterile tips, tubes, etc.

Minimizing the risk of contamination

General practices

Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading).

Clean workspace and sterile tips, tubes, etc.

Minimizing the risk of contamination

General practices

Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading).

Clean workspace and sterile tips, tubes, etc.

If working with difficult samples, such as degraded DNA...

Special laboratory design (for example two separate doors that are opened in sequence, presence of UV light).

Be extra-careful (for example, change gloves more often).

Q&A and discussion

Thank you very much!