InternalLengthStandard

Internal Length Standard for DNA Capillary Electrophoresis

Overview

An internal length standard (ILS, DNA-Ladder, DNA Size Standard) is required for DNA fragment analysis on a capillary electrophoresis (CE) instrument. DNA segments with a known size are usually labelled with a separate fluorescent dye so that they show up in a separate channel of the CE instrument. From comparing the known sizes of the ILS to the unknown peak in an electropherogram it is possible to calculate the exact size of the unknown fragment.

Internal length standards can be purchased from Life Technologies, Promega and other vendors. However they are very expensive. Here we describe a method to create a homemade ILS with a simple PCR assay using just a single fluorescently labeled primer. The template is a highly conserved region of the human mitochondrial DNA where to our knowledge no single insertion or deletion has been observed. Therefore the template DNA can be easily obtained by a cheek swab of a lab person or any other source of human genomic DNA.

Materials

Primers

Primer Name	Sequence	rCRS position	PCR Prod. Length	Tm
ILSmt_3245_F_ROX or BMN5	GCCCGGTAATCGCATAAAAC	3245		61.2 °C
ILSmt_3304_60_R	GTTAAGAAGAGGAATTGAACCTCTGAC	3304	60	61.1 °C
ILSmt_3324_80_R	AGGTTGGCCATGGGTATGT	3324	80	60.1 °C
ILSmt_3344_100_R	TGGGTACAATGAGGAGTAGGAGG	3344	100	61.6 °C
ILSmt_3364_120_R	GAATGCCATTGCGATYAGAATG	3364	120	61.6 °C
ILSmt_3384_140_R	TTTCGTTCGGTAAGCATTAGG	3384	140	59.3 °C
ILSmt_3404_160_R	GTTGTATATRGCCTAGAATTTTTCGTTCG	3404	160	61.8 °C
ILSmt_3424_180_R	AACGTTGGGGCCTTTGC	3424	180	62.0 °C
ILSmt_3444_200_R	AGTAGCCCGTAGGGGCCTA	3444	200	61.0 °C
ILSmt_3469_225_R	TTTTATGGCGTCAGCGAAG	3469	225	60.0 °C
ILSmt_3494_250_R	GTTTTAGGGGCTCYTTGGTG	3494	250	58.7 °C
ILSmt_3519_275_R	TAGAGGGTGATGGYAGATGTGG	3519	275	58.9 °C
ILSmt_3544_300_R	GAGAGCTAAGGTCGGGGC	3544	300	59.9 °C
ILSmt_3569_325_R	GGGTTCATAGTAGAAGDGCGATG	3569	325	59.3 °C
ILSmt_3594_350_R	ACCAGGGGGTTGGGTATG	3594	350	60.4 °C
ILSmt_3619_375_R	AAATAGGAGGCCTAGGTTGAGG	3619	375	60.0 °C
ILSmt_3644_400_R	CGGCTAGGCTAGAGGTGGC	3644	400	62.3 °C
ILSmt_3669_425_R	CACCCTGATCAGAGGATTGAGTAA	3669	425	61.7 °C
ILSmt_3694_450_R	CAGGGCGTAGTTTGAGTTTGAT	3694	450	60.5 °C
ILSmt_3719_475_R	GGGCTACTGCTCGYAGTG	3719	475	60.7 °C
ILSmt_3744_500_R	AGGGTGACTTCATATGAGATTGTTTG	3744	500	61.7 °C
ILSmt_3769_525_R	TAATGTTGATAGTAGAATGATGGCTAGG	3769	525	60.2 °C

The forward primer is labeled with a fluorescent dye. We've used ROX for the red channel or BMN5 for the infrared ("orange") channel. If this ladder is used on a stained agarose gel, then the forward primer can be unlabeled.

For each 50 μL PCR reaction:

• 35 μL H₂O
• 5 μL 10X PCR buffer
• 5 μL 2mM dNTPs (each)
• 1.5 μL 50mM MgCl₂
• 1 μL 50μM ILSmt_3245_F primer, with fluorescent label
• 1 μL 50μM antisense primer
• 1 μL DNA template (human genomic DNA)
• 0.5 μL TAQ DNA polyermerase

Procedure

1. Set up a PCR reaction for each reverse primer in a separate PCR tube.
2. Perform thermocycling program
   1. 95 °C 5 min
   2. 95 °C 30 s
   3. 60 °C 30 s
   4. 72 °C 1 min
   5. Repeat steps 2-4 a total of 30 times
   6. 72 °C 5 min
   7. 15 °C hold

After PCR mix the PCR products in equal ratios. We add double volumes for the 100s bands for easier recognition.

For capillary electrophoresis we use (less than) 0.5 μl ILS mixture per 10 μl formamide each injection.

Notes

In practice it is sufficient to only use the markers for 80, 100, 140, 200, 250, 300, 350, 400, 450, 500 and 525 bases.

ILS with BMN5 label

.

References

Relevant papers and books

1. James W. Schumm, Promega Corporation (1997) Why Use a Size Marker and Allelic Ladders in STR Analysis? - Profiles in DNA 1(2) 11–13

Contact

• Thomas Krahn (thomas (at) tkrahn.com)

or instead, discuss this protocol.