application of genetic analyzer in aflp technique

Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes Aug. 31st- Sept. 4th, 2014

Application of Genetic Analyzer in AFLP Technique

Amr M. Ageez, Ph.D. Genomics facility, AGERI, ARC,

Giza, Egypt.

• Genetic markers.

• Restriction Fragment Length Polymorphism (RFLP).

• Amplified Fragment Length Polymorphism (AFLP).

• Using Genetic Analyzer in AFLP.

• Troubleshooting.

Contents:

Genetic Marker

• Any phenotypic difference controlled by genes, that

can be used for studying recombination processes or

selection of a more or less closely associated target

gene.

• Anything in the genome that is variable and can be

used to compare individuals.

• Detectable allelic variation on a chromosome can be

a phenotype, can also be a unique detectable

sequence of DNA

• Readily detectable sequence of protein or DNA that are

closely linked to a gene locus and/or a morphological or

other characters of a plant

• Readily detectable sequence of protein or DNA whose

inheritance can be monitored and associated with the

trait inheritance independently from the environment

Molecular Marker

Types:

A) protein polymorphisms

b) DNA polymorphisms

DNA-based markers

• Restriction pattern: RFLPs, minisatellites

• PCR amplicon pattern: RAPDs

• DNA sequence: SNP

• Combinations of above: SCARs, SSRs

(microsatellites), CAPs, AFLP

Restriction Fragment Length Polymorphism (RFLP)

• Genomic DNA digested with Restriction Enzymes

• DNA fragments separated via electrophoresis and

transfer to nylon membrane

• Membranes exposed to probes labelled with P32

via southern hybridization

• Film exposed to X-Ray

RFLP Methodology

(Pawlik, 2008)

Origin of polymorphism in RFLP

According to the lanes:

A: Wild type specific pattern.

B: Insertion.

C: Deletion.

D: New Restriction site within the probe site.

E: Loss of restriction site within the probe site.

Origin of polymorphism in RFLP

(Schuler Group, 2008)

– Reproducible

– Co-dominant

– Simple

Advantages of RFLP Technique

1. Need high quality DNA

2. Need to develop polymorphic probes - expensive

3. Relatively slow process

4. Use of radioisotopes limits use to certified laboratories

(non-radioactive labeling systems now in wide use)

5. Large quantities of DNA are needed and procedure is

difficult to automate

Limitation of RFLP Technique

The AFLP™ technique is used to visualize

hundreds of amplified DNA restriction

fragments simultaneously.

AFLP technology combines the power of

restriction fragment length polymorphism

(RFLP) with the flexibility of PCR-based

technology by ligating primer-recognition

sequences (adaptors) to the restricted

DNA.

AFLP: Amplified Fragment Length Polymorphism

Amplified Fragment Length Polymorphism (AFLP)

• Restriction endonuclease digestion of DNA

• Ligation of adaptors

• Amplification of ligated fragments

• Separation of the amplified fragments viaelectrophoresis and visualization

• AFLPs have stable amplification and goodrepeatability

AFLP …1

Genomic DNA digested with 2 restriction enzymes:– EcoRI (6 bp restriction site)cuts infrequently

– MseI(4 bp restriction site)cuts frequently

GAATTCCTTAAG

TTAAAATT

Fragments of DNA resulting from restriction digestion areligated with end-specific adaptors (a different one for eachenzyme) to create a new PCR priming site

Pre selective PCR amplification is done using primers complementary to the adaptor + 1 bp (chosen by the user)

NN N N

AFLP …2

Selective amplification using primers complementary to the adaptor (+1 bp) + 2 bp

NNNNNN NNN NNN

AFLP …3

Sample AFLP Gel

Advantages of the AFLP:

1. Only small amounts of DNA are needed.

2. Unlike randomly amplified polymorphic DNAs (RAPDs) that use multiple,

arbitrary primers and lead to unreliable results, the AFLP technique uses

only two primers and gives reproducible results.

3. Many restriction fragment subsets can be amplified by changing the

nucleotide extensions on the adaptor sequences. Hundreds of markers

can be generated reliably.

4. High resolution is obtained because of the stringent PCR conditions.

5. The AFLP technique works on a variety of genomic DNA samples.

6. No prior knowledge of the genomic sequence is required.

What is new?

Genomic DNA digested with 2 restriction enzymes:

Adaptors Ligation

Pre selective PCR amplification

NN N N

Selective amplification

NNNNNN NNN NNN

nine EcoRI fluorescent dye-labeled primers and nine unlabeled MseI primers

Machines

PE 310 Single Capillary System

Technology of Capillary Electrophoresis

PE 310 Single Capillary System

Genetic Analyzer 3100 Capillary System

AFLP Electropherogram

Source: Wikimedia Commons

Peak Height

Fragment Size (bp)

AFLP Fluorescent electrophoresis

Tomato AFLP samples showing Mendelian segregation

The overlapping electropherograms in the panel are AFLP results of sample DNA from three

individuals: parent one (P1), parent two (P2), and F1 from a cross. A and B are the two

significant peaks on this panel and appear only in P2 and F1. GeneScan Reference Guide, LifeTech.

Genome size and AFLP

AFLP Core kit

In the microbial genomes targeted by the AFLP Microbial Fingerprinting Kit, the

core primer sequence is used. In larger genomes, such as plants and some fungi,

this amplification would create too many fragments. In those cases, the

preselective amplification is performed with additional nucleotides on the end of

each primer. Each added nucleotide reduces the number of sequences by a

factor of four

• Additional PCR amplifications are run to reduce the

complexity of the mixture further so that the fragments can be

resolved on a polyacrylamide gel. These amplifications use

primers for the selective amplification of the products. After

PCR amplification with these primers, a portion of the

samples is analyzed on a Applied Biosystems DNA

Sequencer

• The sequences of the adaptors and the restriction site serve

as primer binding sites for a subsequent low-level selection

or “preselective” amplification of the restriction fragments.

The MseI complementary primer contains a 3´ C. The EcoRI

complementary primer contains a 3´ A (Regular Plant

Genome Kit modules, lifeTech.) or no base addition (Small

Plant Genome Kit modules, LifeTech.).

examples of AFLP fingerprint patterns that were prepared using different selective

primers. Note that the EcoRI selective primers with one-nucleotide extensions

(EcoRI-A, EcoRI-T, and EcoRI-G) give simpler patterns than that obtained using the

primer with no extra nucleotide (EcoRI-0).GeneScan Reference Guide, LifeTech.

GeneScan Reference Guide, LifeTech.

GeneScan Reference Guide, LifeTech.

unacceptable primer combinations

Troubleshooting

A. Troubleshooting PCR Amplification

Problems with Poor AmplificationFaint or no signal from sample DNA and from positive control

• Insufficient enzyme in reactions

• Incomplete activation of AmpliTaq Gold™ DNA Polymerase

• Too little sample DNA added to reaction

• Incorrect or suboptimal thermal cycler Parameters

• Tubes not seated tightly in the thermal cycler during amplification

• PCR Master Mix not well mixed before Aliquoting

• Primer concentration too low

• Primers degraded

• Too little free Mg2+ in reaction

• Incorrect pH

Troubleshooting


Problems with Poor AmplificationGood signal from positive control but faint or no signal from sample DNA

• Sample contains PCR inhibitor (for example, EDTA, or certain dyes)

• Pipetting errors.

• Sample DNA is degraded

• Insufficient sample DNA added because of

• inaccurate quantitation

• Primer choice not optimal (for example, primers may be annealing to sites of

template secondary structure or may have internal secondary structure).

• Tm of primers is lower than expected.

Troubleshooting


Problems with Poor AmplificationPoor yield for multiplex PCR

• Non-optimal thermal cycling parameters

• Competition from mispriming and other competing side reactions

• Problems with primer choice, concentration, or degradation

Yield gets progressively poorer for successive PCR amplifications performed over time• Expired or mishandled reagents

Inconsistent yields with control DNA

• Combined reagents not spun to bottom of PCR sample tube.

• Combined reagents left at room temperature or on ice for extended periods of time

• Combined reagents not thoroughly mixed

• Pipetting errors.

Troubleshooting


Extra peaks appear with no discernible pattern

• Presence of exogenous DNA

• Nonspecific priming

• Primer-dimer and primer-oligomer artifacts

• Incomplete restriction (and/or ligation if performing AFLP)

• Too much DNA in reaction so that insufficient adaptor is present

• Mixed sample

Troubleshooting

B. Troubleshooting Genetic Analyzer

Data was not automatically analyzed

• Sample Sheet not completed or completed Incorrectly

• Injection List not completed or completed Incorrectly

• Analysis preferences set incorrectly in data collection program

• Insufficient free RAM

Low current

• Small bubble in capillary blocking current flow

• Small bubble in pump block

• Plugged, broken, or no conducting capillary

• Poor quality water in buffer solutions

Troubleshooting


No current

• Too little or no buffer in anode buffer reservoir

• Electrode bent

• Capillary bent away from electrode

• Unfilled capillary or bubbles in capillary

• Major leaks in system. Polymer does not enter capillary

• Pump blockage (pump is plugged with urea or crystallized buffer)

• Anode buffer valve does not open

• Plugged, broken, or no conducting capillary

• Poor quality water in buffer solutions

• Incorrect polymer solution formulation

• Corrupted firmware

• Syringe Pump Force too low. Capillary is not being filled completely

Troubleshooting


Current too high

• Decomposition of urea in polymer solution

• Incorrect buffer formulation (most likely too concentrated)

• Arcing to conductive surface on the instrument

Troubleshooting


Problems with Peak Resolution

• Poor capillary performance

• Incorrectly prepared and/or old buffer or polymer solutions

• Injection time too long (broad peaks)

• Incorrectly prepared and/or degraded sample

• Incorrect buffer formulation, or polymer composition

• Electrophoresis voltage too high.

• Sample concentrated by evaporation leaving excess salt behind.

• Incomplete strand separation due to insufficient heat denaturation

• Too much DNA in sample

• Poor quality water

• Capillary too short

Troubleshooting


Problems with Signal Strength and Quality

No signal

• No sample added

• Sample not at bottom of tube

• Air bubble at bottom of sample tube

• Capillary misaligned with electrode

• Capillary bent out of sample tube

• Autosampler not calibrated correctly

• Sealed sample tube septum

Troubleshooting



Too low signal

• Insufficient sample added

• Samples added to formamide that has degraded to formic acid and formate ions

• Ions in sample

• Sample not thoroughly mixed with formamide/size standard mixture

• Insufficient [F]dNTPs added to PCR reaction

Too high signal

• Too much sample injected into capillary

• Unincorporated [F]dNTPs

Troubleshooting



High baseline

• Dirty capillary window

• Capillary moved out of position in front of laser window

• Precipitate in polymer

• Incorrectly prepared and/or old buffer or polymer solutions

• Defective capillary

• Matrix made incorrectly resulting in too much correction (also indicated by troughs

under peaks)

Thank you

application of genetic analyzer in aflp technique

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