IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons create a binding between genomic DNA and their conserved ends. To identify
polymorphisms for retrotransposon insertion, marker systems rely on PCR amplification between these ends
and some components of flanking genomic DNA.

REMAP: Amplification between retrotransposon proximal to simple sequence repeats (microsatellites)

producing the marker bands.

IRAP: Products are generated from two nearby retrotransposons using outward-facing primers.

PROTOCOL

Adapted from the original paper. https://www.nature.com/articles/nprot.2006.377

Materials

Reagents

1 Ethidium bromide (diluted in H20) 0.5mg ml-1

2 DNA ladder (Electrophoresis)
100-10000 pb or similar
Goal:
GeneRuler DNA ladder mix cat. No. SM1173, Fermentas
(Discontinued and redesigned by Thermo)
https://www.fishersci.es/shop/products/fermentas-
generuler-dna-ladder-mix-1/11883963 Thermo
Scientific™ SM0332

 Do not heat the DNA ladder before loading.

 Load the same volumes of the DNA sample and the ladder onto the gel.
 For quantification, adjust the concentration of your DNA sample so that your band of interest is
approximately matched in mass (ng) to the nearest band of the ladder.

Equipment

1 Power supply for Electrophoresis Minimum: 300 V 400 mA

2 UV transilluminator (for visualization of Viewing area: 20 x 20 cm
ethidium bromide-stained gels)

Reagents Setup

Agarose

1 Resolute wide Range (cat. No. 337100, BIOzym) Discontinued

2 SERVA Premium (cat. No. 11381)

High-purity agarose for analytical and preparative gel

electrophoresis of DNA/RNA fragments with a wide https://www.serva.de/enDE/
separation range between 500 bp and >20,000 bp. ProductDetails/
48_11381_Agarose_SERVA_Premium_mo
Low gel background, low DNA binding, and low EEO lecular_biology_grade_213_0.html
enables high-resolution separation, even at higher gel
 concentrations.

Gene technology grade, suitable for molecular biology

methods such as blotting, cloning, restriction enzyme
analyses, sequencing, recovery of DNA fragments for
further modifications (restriction analysis, ligation
reactions).
Agarose MP (Cat no 1091.0250) AppliChem:

DNases/RNases/Proteases: not detectable Ash: ≤ 0.25

% Electroendoosmosis (EEO): ≤ 0.12
Moisture: ≤ 7 % https://www.itwreagents.com/italy/en/
Gel point: 36 ± 1.5°C product/agarose+mp/A1091
Gel strength (1 %): ≥ 1800 g/cm2
Gel strength (1.5 %): ≥ 3200 g/cm2
Melting point: 88 ± 1.5°C Sulfate: ≤ 0.12 % WGK: 1

TopVision CG Agarose (Cat. No. R0491, Fermentas).

Thermo Scientific TopVision Agarose provides optimal

concentration between 0.7 to 2% in all typical buffer
systems. It is a highly purified agarose with very low
EEO values certified by strict quality control test
procedures.

Highlights

• Optimal concentration between 0.4 to 5% in all typical

buffer systems https://www.thermofisher.com/order/
• GQ (Genetic Quality) certified ensures that nucleic catalog/product/R0491
acids recovered from preparative gels can be used for
downstream applications (enzymatic reactions, etc.)
• Low DNA/RNA binding

Product Type Specs

Separation Range: 100 bp to >30 kb
Melting Point: 88 °C
Product Line: TopVision
Validated Application: Gel Electrophoresis

NOTE ABOUT AGAROSES

 1% agaroses with gel strength > 1700 g cm-2 can be used.

L Agaroses LE are not effective for fine resolution of fingerprinting bands.
L Cambrex and Metaphor Agaroses have low gel strength and make for difficult gel manipulation.

Loading buffer
Prepare 10x buffer:
1. 30% (wt/vol) Ficoll 400 (Sigma)
2. 100mM Tris-HCL (Ph 8.0)
3. 10Mm EDTA
4. 0.01% WT/VOL Orange G
5. 0.01% Xylene Cyanol FF in mili-Q (Millipore Ultrapure deionized water)
 6. 0.4M Tris H3BO3
7. 0.1 M Na2B4O7
8. 20 mM EDTA
Ph 8.6 in Milli-Q Ultrapure deionized water.  It also brings better results with STBE, standard
TAE and TBE buffer.

SBTE electrophoresis buffer

Prepare 20x SBTE:

1. 0.4 M Tris H3BO3

2. 0.1 M Na2B4O7
3. 20Mm EDTA
pH: 8.6 in Milli-Q Ultrapure deionized water

Note: To get better results, use STBE

Standard TAE and TBE also work.

PCR Protocol

One Taq 2X Master Mix with Standard buffer 100rx (3) New England M0482S
Protocol: https://international.neb.com/protocols/2012/09/06/protocol-for-onetaq-2x-master-mix-
with-standard-buffer-m0482

Reaction setup from New England Biolabs:

We recommend assembling all reaction components on ice and quickly transferring the reactions to a
thermocycler preheated to the denaturation temperature (94°C).

1. Add to a sterile thin-walled PCR tube:

2. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
Overlay the sample with mineral oil if using a PCR machine without a heated lid.
3. Transfer PCR tubes to a PCR machine and begin thermocycling:
Primers:

Oligo: Sukkula:

Seq: 5’-GATAGGGTCGCATCTTGGGCGTGAC-3’ (25mer)

Oligo: Nikita:

Materials Checklist

Availability Product Expiration

n Product available date
Yes No Name it
1 Ethidium bromide
2 DNA Ladder 100-10000 bp
3 Power supply min. 300v 400mA for Electrophoresis
4 Horizontal electrophoresis apparatus
Medium or large scale
5 Gel comb (36 wells – 1.0 mm thickness – 4.0mm wide
wells- 1.0mm wide well spacing.
6 Agarose (Not LE or Metaphor)
7 Ficoll 400 powder
8 Tris-HCL
9 EDTA
10 Orange G
11 Xylene Cyanol
 12 Milli-Q Millipore ultrapure deionized water
13 Tris
14 Boric Acid (H3BO3)
15 Tris- H3BO3 (In case the Boric Acid is not available)
16 Sodium Tetraboratum (Na2B4O7) (Borax)
17 Taq Polymerase
18 PCR Reaction buffers (in case Taq is not ‘‘One Step’’
19 Thermal cycler
 Requiring: Rapid heating-colling system (4°C to 99°C)
 Temperature change by 3°C per second

20 Digital gel electrophoresis scanner (Resolution 50-100µm)

21 Functional Software for electrophoresis reading
22 Computer for electrophoresis reading

THEORY OF DNA FINGERPRINTING ELECTROPHORESIS

DNA fingerprinting allows the comparison of DNA from different organisms and the identification of a
particular individual. Basically, DNA is extracted and cut into fragments using restriction enzymes. The
fragments form a pattern on agarose gel electrophoresis according to their length. The longer the DNA
fragments are generated, the larger their molecular weight and the shorter they travel in an electrophoresis
setting. This pattern looks a lot like the barcode on products in the supermarket and it resembles the
individual’s DNA fingerprint.
To interpret the pattern formed, one must realize that restriction enzymes cut the DNA at specific base-pair
sequences called recognition sequences. There are hundreds of restriction enzymes, each having a specific
recognition sequence made of four to twelve base pairs. The lengths of the fragments generated by a
restriction enzyme digest of an extracted DNA sample depend upon the number of cuts made and their
locations. This depends on the number of recognition sequences of the enzyme used on the extracted DNA
and their locations. Thus, everyone’s DNA is cut by restriction enzymes into distinctive different sized
fragments that appear on the electrophoresis gel in the form of bands.

Principle of Work

Agarose is isolated from the seaweed genera Gelidium and Gracilaria. It’s mixed with a buffer and heated in a
microwave, then left to cool down before pouring in the cast. A comb is added at a specific site to form the
wells required for sample upload. Then the gel is left to solidify.
The concentration of gel = weight of agarose/volume of buffer (g/ml). For a standard agarose gel
electrophoresis, 0.8% gel gives good separation of large 5–10kb DNA fragments, while 2% gel gives a good
resolution for small 0.2-1 Kb DNA fragments.
During gelation, agarose polymers associate non-covalently and form a network whose pore sizes determine a
gel's molecular sieving properties.

The phosphate in the backbone of DNA is negatively charged, therefore DNA fragments will migrate to the
positively charged anode.
DNA has a uniform mass/charge ratio, therefore DNA molecules are separated by size within an agarose gel
in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight.
The rate of migration of a DNA molecule through a gel is determined by the following:
1) size of DNA band (the heavier, the slower)
2) agarose gel concentration (usually 0.8%)
3) DNA conformation (linear/plasmid/etc..)
4) Voltage
5) Electrophoresis buffer.
6) Ethidium bromide: EtBr is positively charged, thus; reducing the DNA migration rate by 15%. Other stains
for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue.

UV light activates electrons in the aromatic ring of ethidium bromide releasing light as electrons return to the
ground state. EtBr intercalates itself in the DNA molecule in a concentration-dependent manner. So higher
intensity means a higher amount of DNA.

Restriction enzymes are present in bacteria. They are named according to the bacteria from which they are
extracted. They protect bacteria by digesting foreign DNA at specific sequences.
Restriction enzymes such as (EcoRI) and (HindIII) are used in this experiment to Cut the extracted DNA.
EcoRI recognizes the 6 bp sequence 5’ GAATTC 3’ and makes a staggered cut between the G and A creating
sticky ends. HindIII recognizes the 6 bp sequence 5’ AAGCTT 3’ and makes a staggered cut between the A
and A creating sticky ends. Both enzymes cut best at 37°C.

EcoRI:
5’ G^AATTC 3’
3’ CTTAA^G 5’

HindIII:
5' A ↓AGCTT 3'
3' TTCGA ↑A 5'