Hair and Nail DNA Extraction Kit

ISOHAIRthManual (11 edition)
Code No. 315-03403

for 10 extractions

Code No. 319-03401

for 100 extractions

NIPPON GENE CO., LTD.

Table of contents

I

Product description

1

II

Contents of kit

1

III Storage

1

IV Protocol

2

V

Troubleshooting

5

VI Data collection

6

1. Examples of experiments using human hair,

6

nail and mucous membrane of oral cavity
2. Examples of experiments using human hair
VII References

7
12

Precautions○ This product is a reagent for research and cannot be used for medical or otherobjectives. Also, this product should be handled only by persons having a basic
knowledge of reagents.
○ Handle this product in accordance with the descriptions in the manual. We
are not responsible for problems caused if this product is not handled inaccordance with the manual.

ISOHAIR Manual (ver.11)
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I

Product description

DNA extraction from human hair and nail is a very important technique in the field of
forensic medicine. Allowing simple, rapid DNA extraction from such samples is highly
valuable compared to the conventional test in which blood or tissue samples are utilized.
DNA extraction from hair and nail is also highly useful for its unnegligible characteristics:
easy sampling and tolerance to viral infection from the conventional samples in the fieldof molecular biology.
ISOHAIR is a kit for DNA extraction from human hair and nail.
The major component of hair and nails is keratin which is a protein difficult to decompose.
In order to dissolve hair completely, it normally requires incubation in a period of longtime in a buffer containing a protein degrading enzyme.
Complete degradation of hair can be achieved in about 30 min using this kit. This kit
further enables simple, rapid DNA extraction from hair in about 1 hr in an efficient way
especially when human genomic DNA is urgently needed in a short period of time.
ISOHAIR is also applicable to DNA extraction from mouse hair or nail.

II

Contents of kit*1

Extraction Buffer
Enzyme Solution
Lysis Solution
Ethachinmate
3 M Sodium Acetate (pH 5.2)
TE (pH 8.0)

(For 100 extractions) (For 10 extractions)
20 ml
1 ml × 2
1 ml
100 μl0.8 ml80 μl
0.2 ml20 μl
2 ml
200 μl1 ml × 2
200 μl*1 White crystals may appear in the Extraction Buffer but this will not affect the quality.
In that case, ensure to dissolve the crystals in a water bath about at 50°C before use.
The Extraction Buffer contains a protein denaturant and should be handled with care.
If the product enters eyes or adheres to skin, immediately wash well with large
amounts of water.
This product does not include phenol/chloroform/isoamyl alcohol, ethanol, primers andthe like.

III

Storage－20℃-1-

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IV

ProtocolHair *1)
Wash with ethanol *2)
Cut hair sample into 4-5 mm pieces with scissors or the like.
← Extraction Buffer 200 μl *3)← Enzyme Solution 5 μl
← Lysis Solution 8 μl
Mix *4)
Incubate, 55°C, 20 min *5)← Enzyme Solution 5 μl
Mix *4)
Incubate, 55°C, 5-10 min *6)← Phenol/chloroform/isoamyl alcohol (25:24:1) 200 μl
Mix by inversion *7), 5 min
11 K × g, room temperature, 5 min
Aqueous phase← 3 M Sodium Acetate (pH 5.2) 20 μl← Ethachinmate 2 μl *8)
Mix *4)← Ethanol 400 μl *9)
11 K × g, room temperature, 15 min
Precipitate← 70％ ethanol 1 ml
Wash by gentle mixing by inverting several times.
Precipitate
Dry *10)← TE (pH 8.0) 20 μl
DNA solution *11)

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*1) Required volume of sample is as follows.
Human hair: 1 cm hair root sample cut at 1 cm distance from the root end OR
4 cm hair shaft sample cut at distances of 2 cm and 6 cm from the root end.
[See VI. “Amount of DNA extracted from hair” (p. 10)]

Human nail: 2 pieces of nail tip cut into 1 x 1mm squares (total of 0.5 mg)
Mouse body hair: a pinch of mouse hair (about 5 mg) cut off with scissors.
Mouse nail: 1-2 mm mouse nail tip
Using of fresh samples is recommended to obtain high DNA yields. [See VI. “Elapsed
time after hair removal and DNA yield” (p. 11)]*2) Hair cosmetics may inhibit PCR reaction. Wash hair samples thoroughly withethanol as follows before proceeding to the next step when they are adhered with haircosmetics, dirt, or dust.
(1) Add about 1 ml of ethanol to a 1.5 ml plastic tube.
(2) Put uncut hair sample into (1) using forceps.
(3) Invert (2) several times.
(4) Take out the sample with forceps, place on filter paper or the like and remove
as much ethanol as possible.
If a hair sample is clean and if it is not adhered with foreign subjects, there is no need
to wash with ethanol. It is also recommend that mouse body hair is washed withethanol before starting the procedure. When using a manicured nail sample, remove
manicure paint first.
*3) White crystals may appear in the Extraction Buffer but this will not affect the quality. In
that case, ensure to dissolve the crystals in a water bath at about 50°C before use
and to homogenize by stirring.
The Extraction Buffer contains a protein denaturant and should be handled with care.
If the product enters eyes or adheres to skin, immediately wash well with large
amounts of water.
Perform all of the following steps before ethanol precipitation at room temperatureunless otherwise noted.
*4) Mix by tapping tube several times with a finger.
vigorous mixing such as vortexing.

Avoid*5) Hair starts degrading during this incubation
Reaction temperature can be 37°C or room temperature.
The reaction time at 37°C is 2-3 times longer than at 55°C,
and 3-5 times longer at room temperature.
*6) Small pieces of digested hair sample deposit at the bottomof tube during dissolution step.

After *5)

After *6)
(Complete dissolution)-3-

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Mix by tapping the bottom of the tube gently with a finger every 2-3 min during thereaction. Dissolution time varies depending on the type and volume of the hair.
If undigested pieces of hair remain after incubation (after *6)), add 5 μl of the Enzyme
Solution and incubate at 55 °C for 5-10 min again. If the hair still cannot be dissolved
completely even after second incubation with additional Enzyme Solution, continue
the procedure with the obtained solution as it should contain eluted DNA.
NOTE: mouse hair normally cannot be dissolved completely.
In the case of using human or mouse nail sample, there is only a little visible change
in appearance (or seemingly no change) but continue the procedure with theobtained solution as it should contain eluted DNA.
*7) Mix gently by inversion. Avoid vigorous mixing such as vortexing.
*8) Addition of Ethachinmate during ethanol precipitation enhances efficiency in DNA
extraction. For the feature of Ethachinmate, it can accelerate the DNA extraction
process by omitting incubation at -20°C in the conventional ethanol precipitation.
*9) When the final DNA yield is low, it may be increased by incubation at -20°C for 30 min
after the step 9).
*10) Air dry or briefly vacuum dry pellet for 5-10 min. It is important not to over-dry thepellet as it may become harder to resuspend.
*11) When DNA is extracted from a hair root sample, there is a risk of RNA contamination.
Treat the solution with RNase to eliminate RNA as necessary.

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V Troubleshooting● Hair sample does not dissolve.
・After the step *6) , add another 5 μl of the Enzyme Solution and incubate at
55°C.
・Even if hair cannot be dissolved completely, continue the procedure with theobtained solution as it should contain eluted DNA.
● No band or faint band in gel electrophoresis.
・Amount of DNA extracted from hair varies between individuals as well as
between individual hair strands from the same person. Even in an identicalhair sample, it also varies in sample types of hair: hair root and shaft. Since
only a small amount of DNA can be extracted from a hair sample (especiallyfrom hair shaft), a band may not be observed on a gel after electrophoresis. In
that case, it is recommended to proceed to the PCR step. → See VI. “Amountof DNA extracted from hair” (p. 10).
● Colored DNA solution
・The dye melanin contained in hair may have eluted out. Melanin is known to
inhibit PCR reaction but addition of T4 gene 32 protein may moderate theinhibition. →See VI 2. “Moderation of PCR inhibition using T4 gene 32
protein” (p. 8, 9).
● No amplification in PCR.
・Use a hair root or a part that is as close to the hair root as possible. → See VI.
“Position in hair and amount of DNA” (p. 10).
・Use a fresh sample. → See VI. “Elapsed time after hair removal and DNA yield” (p.
11)
・If the obtained DNA solution is colored, it may contain the PCR inhibitor melanin.
Add T4 gene 32 protein to moderate the inhibition. → See VI. “Moderation of
PCR inhibition using T4 gene 32 protein” (p. 8, 9).
・Optimize PCR conditions.
Change the primer design, increase the amount of Taq DNA polymerase,change the denaturing temperature, etc.
● Smeared PCR amplified product.
・Reduce the amount of Taq DNA polymerase.
・Increase the amount of the template.
・Set the denaturing temperature higher.
・Make the primers longer. → See VI. “PCR condition” (p. 11)● Nonspecific amplification.
・Change the primer design.
・Extract after washing hair with ethanol thoroughly.

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VI Data collection
1. Examples of experiments using human hair, nail and mucous
membrane of oral cavity
DNA was extracted using the ISOHAIR from hair root, nail, and mucous membrane of theoral cavity.
Oral mucous membrane sample was collected with a cotton-tipped swab and DNA
sample was collected in different methods: (1) collecting pellet after the sample wassuspended in 1 x PBS and centrifuged; (2) forming suspension by directly inserting thecotton-tipped swab into the Extraction Buffer. (See the Nippon Gene website for moredetailed experimental procedures).
Electrophoresis was performed using 1/4 of amount of DNA obtained from each sample.

Lane 1:
Lane 2:
Lane 3:
Lane 4:Lane 5:
Lane 6:
Lane 7:
Lane 8:Lane 9:MarkerHair root, 1 cm ×3
Nail, 1 mm cube ×2
Oral mucous membrane (1)Oral mucous membrane (2)Control: cotton swab (1)Control: cotton swab (2)Negative control
MarkerMarker 1 (λ/Hind III digest)
0.8% Agarose S

Human p53 gene (Exon 10; 279 bp) was amplified by PCR using 1/10 of amount of DNA
solution obtained from each sample and electrophoresed.
＜PCR mixture＞
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol /μl)
Gene Taq NT (5 units/μl)
H2O
Total
＜PCR condition＞
94°C 1 min.
94°C 30 sec.
55°C 30 sec.
72°C 1 min.
72°C 5 min.

Marker 5 (φX174/Hinc II digest)
3% Agarose 21

-6-

2 μl
5 μl
4 μl
1 μl
1 μl
0.5 μl36.5 μl
50 μl

35 cycles

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2. Examples of experiments using human hair
Detection of mitochondrial DNA
Using the ISOHAIR, DNA was extracted from 1 cm hair root and 6 cm hair shaft fromthree different human subjects (A, B, C). Using 1/4 amount of the obtained DNA, humanmitochondrial DNA (D loop region; 280 bp) was amplified by PCR and electrophoresed.
M

Hair rootA
B
CA

Hair shaft
B
C＜PCR mixture＞
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer (20 pmol each/μl)
Gene Taq NT (5 units/μl)
H2O
Total
＜PCR condition＞
94°C 1 min.
98°C 15 sec.
55°C 15 sec.
72°C 30 sec.
72°C 5 min.

M: Marker 5 (φX174/Hinc II digest)
3% Agarose 21

5 μl
5 μl
4 μl
1 μl
0.5 μl34.5 μl
50 μl

40 cycles

Detection of p53 gene
DNA was extracted from 1 cm hair root and 6 cm hair shaft using the ISOHAIR. Using 1/4
amount of the obtained DNA, the p53 gene (exon 11) was amplified by semi-nested PCR(first PCR product 1,296 bp, second PCR product 265 bp)1) and electrophoresed.
first＜PCR Mixture＞
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
H2O
Total

Hair shaft

Hair root

Hair shaft

Hair rootMsecond

M: Marker 4 (φX174/Hae III digest)
3% Agarose 21

Using the first PCR product amplified from thehuman p53 gene, cycle sequencing was
performed, and sequencing for both hair root andhair shaft was observed successfully.

-7-

5 μl
4 μl
1 μl
1 μl
0.25 μl
50 μl＜first PCR condition＞* 5 μl was used as a template.
94°C 1 min.
98°C 15 sec.
55°C 15 sec.
40 cycles
72°C 30 sec.
72°C 5 min.
＜second PCR condition＞* 1 μl of the first PCR product was
94°C 1 min.
used as a template.
98°C 15 sec.
60°C 15 sec.
30 cycles
72°C 30 sec.
72°C 5 min.

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Relief of PCR inhibition with T4 gene 32 protein
DNA solution extracted from hair may have a dark brown or black coloring. This isespecially conspicuous when DNA is extracted from the tip end of hair that is 15 cm or
longer, or bleached hair. The causative substance is considered to be the dye melanincontained in hair which is reported to inhibit PCR3).
Using the ISOHAIR, DNA was extracted from hair with high melanin content.
To the reaction mixture, for which PCR amplification had been already confirmed (Lane1), 1/20 amount of the obtained DNA was added, and no amplification was observed(Lane 2). T4 gene 32 protein was then added to the mixture, and amplification was
successfully observed (Lane 3).
Also, no amplification of human mitochondria DNA (280 bp) was observed (Lane 4) in
PCR using the obtained DNA as a template, but amplification was observed when T4
gene 32 protein was added (Lane 5).
M

1

2

3

4

5

Lane 1:
Lane 2:

Amplification of 900 bp using ColE1 as a template
Lane 1 + 1/20 of the amount of 6 cm hair shaft DNA
containing a significant amount of melanin← 900 bp

Lane 3:
Lane 4:← 280 bp

Lane 2 + T4 gene 32 protein (2 μg)
Amplification of human mitochondrial DNA (280 bp)
using as a template 1/20 of the amount of DNA
containing a significant amount of melanin extracted
from 6 cm hair shaft.

M: Marker 5 (φX174/Hinc II digest)
3% Agarose 21

Lane 5:＜ColE1 PCR mixture＞

Lane 4 + T4 gene 32 protein (2 μg)＜Human mitochondria DNA PCR mixture＞

Template DNA (ColE1/Sau96 I digest)
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
Total (H2O to volume)

0.1 ng
5 μl
4 μl
1 μl
1 μl
1 μl
40 μl

10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
H2O
Total

5 μl
4 μl
1 μl
1 μl
1 μl
28 μl
40 μl

Lane 1

Lane 2

Lane 3

Lane4

Lane5

ColE1 PCR mixture

40 μl

40 μl

40 μl

Human mitochondria DNA PCR mixture

40 μl

40 μl

DNA solution with much melanin

0 μl

1 μl

1 μl

DNA solution (template) with much melanin

1 μl

1 μl

T4 gene 32 protein (0.5 μg/μl)

0 μl

0 μl

4 μl

T4 gene 32 protein (0.5 μg/μl)

0 μl

4 μl

H2O

10 μl

9 μl

5 μl

H2O

9 μl

5 μl

Total

50 μl

50 μl

50 μl

Total

50 μl

50 μl

＜PCR condition＞
94°C 1 min.
94°C 20 sec.
55°C 20 sec.
72°C 20 sec.
72°C 5 min.

＜PCR condition＞
98°C 1 min.
98°C 15 sec.
60°C 15 sec.
72°C 30 sec.

20 cycles

72°C

-8-

35 cycles

5 min.

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<Reference data>
Examples are shown to indicate the relationship between the application volumes ofmelanin and of T4 gene 32 protein.
Relief of PCR inhibition by melanin with T4 gene 32 protein is described in the previous
page, but T4 gene 32 protein itself can inhibit PCR reaction if an excessive amount is
added.
Also, in the case of very high melanin content, it is expected that the addition of T4 gene
32 protein cannot relief the inhibition.
Melanin 0 ng
T4 gene 32 protein (μg)
M

0 0.1 0.5 1

2Melanin 150 ng

5 10

0 0.1 0.5 1

2

5

10

M: Marker 2(λ/Hind III・EcoR I double digest)
0.8% Agarose S

Melanin 15 ng
T4 gene 32 protein (μg)
M 0 0.1 0.5 1 2 5 10

Melanin 300 ng
0 0.1 0.5 1＜ColE1 PCR mixture＞
Template DNA (ColE1/Sau 96 I digest)
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
MelaninT4 gene 32 protein
H2O
Total (H2O to volume)＜PCR condition＞
94°C 1 min.
94°C 20 sec.
55°C 20 sec.
72°C 1 min.

2

Melanin 600 ng
T4 gene 32 protein (μg)
M 0 0.1 0.5 1 2 5 10

5 10

0.1 ng
5 μl
4 μl
1 μl
1 μl
1 μl
0, 15, 150, 300, 600 ng
0, 0.1, 0.5, 1, 2, 5, 10 μg
50 μl

20 cycles-9-

ISOHAIR Manual (ver.11)
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Amount of DNA extracted from hair
Using the ISOHAIR, DNA was extracted from hair roots or hair shafts of three humansubjects (A, B, C), and electrophoresis was performed.
The results indicate that DNA yield varies between individuals and even between hairstrands from the same individual. Based on the result of gel electrophoresis, the amountof extracted DNA is estimated to be about 0.5 μg per hair root and less than 10 ng per
hair shaft. 4).
Ｍ

1

2

3

4

Ｍ

5

6

7

8

9

←＊

Lane 1: Hair root A
Lane 2: Hair root A
Lane 3: Hair shaft A
Lane 4: Hair shaft A
Lane 5: Hair shaft A
Lane 6: Hair root B
Lane 7: Hair root B
Lane 8: Hair root C
Lane 9: Hair root C

1 cm
1 cm×5
6 cm
18 cm
36 cm
1 cm
1 cm×5
1 cm
1 cm×5

M: Marker 2* The lower bands of Lane 1, 2, 7 (indicated by arrow) are RNA.

(λ/Hind III・EcoR I double digest)

0.8％ Agarose S

Regional difference of hair in DNA yield
DNA yield varies depending on a region of hair sampled even for the same length of hair.
Using the ISOHAIR, DNA was extracted from each region of 0-6 cm, 6-12 cm, 12-18 cm,
18-24 cm, 24-30 cm, 30-36 cm and 36-42 cm from the root end of hair with total lengthsof 45 cm and 11 cm. Using 1/4 amount of the obtained DNA, human mitochondrial DNA(D loop region; 280 bp) was amplified by PCR, and electrophoresed.
Increasing trend in amount of amplified DNA was seen in the sites closer to the root end,suggesting that more DNA is contained in samples closer to the hair root.

Total lengths of 45 cm

M

11 cm

0

36

30

24

18

12

6

0

6

6

42

36

30

24

18

12

6

11

(cm)

＜PCR mixture＞
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer (20 pmol each/μl)
Gene Taq NT (5 units/μl)
H2O
Total
＜PCR condition＞
94°C 1 min.
98°C 15 sec.
55°C 15 sec.
72°C 30 sec.
72°C 5 min.

M: Marker 5 (φX174/Hinc II digest)
3% Agarose 21- 10 -

5 μl
5 μl
4 μl
1 μl
0.5 μl34.5 μl
50 μl

40 cycles

ISOHAIR Manual (ver.11)
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Elapsed time after hair removal and DNA yield
Amount of DNA that can be extracted from hair gradually decreases over time after theremoval.
Using ISOHAIR, DNA was extracted from 4 cm hair shaft samples that were left standing for
76 days, 57 days, 31 days and 0 days. Using 1/4 amount of the obtained DNA, humanmitochondrial DNA (D loop region; 280 bp) was amplified by PCR and electrophoresed.
Larger amount of amplified DNA was extracted from a hair sample with shorter time after
removal, suggesting either a higher yield or less elution of the PCR inhibitor.

M

76

57

31

0

M: Marker 5 (φX174/Hinc II digest)
3% Agarose 21(Days)＜PCR mixture＞
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)primer-forward (20 pmol/μl)
primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
H2O
Total
＜PCR condition＞
98°C 1 min.
98°C 15 sec.
60°C 15 sec.
72°C 30 sec.
72°C 5 min.

5 μl
5 μl
4 μl
1 μl
1 μl
0.5 μl33.5 μl
50 μl

35 cycles

PCR condition
Since only a small amount of DNA can be extracted from a hair sample, it is significant toobtain optimal PCR conditions in order to improve reproducibility.
Using the ISOHAIR, DNA was extracted from 1 cm of hair root and 6 cm of hair shaft.
Human mitochondrial DNA (D loop region; 280 bp) was amplified by PCR using 1/4 amount of
the obtained DNA as a template and 20 mer or 30 mer primer and electrophoresed. The result
indicates that nonspecific amplification was inhibited with the longer primer.

M

Hair root

Hair shaft

20

20

30

30 (mer)

M: Marker 5 (φX174/Hinc II digest)
3% Agarose 21<PCR mixture>
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)
20 mer primer-forward (20 pmol/μl)
20 mer primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
H2O
Total
Template DNA
10×Gene Taq Universal Buffer
dNTP mixture (2.5 mM each)
30 mer primer-forward (20 pmol/μl)
30 mer primer-reverse (20 pmol/μl)
Gene Taq NT (5 units/μl)
H2O
Total

- 11 -

5 μl
5 μl
4 μl
1 μl
1 μl
0.25 μl＜PCR condition＞
94°C 1 min.
98°C 15 sec.
55°C 15 sec. 40 cycles
72°C 30 sec.
72°C 5 min.

33.75 μl
50 μl
5 μl
5 μl
4 μl
1 μl
1 μl
0.25 μl33.75 μl
50 μl

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Other experimental examples are also shown under “ISOHAIR” on the product page of
the Nippon Gene website.
List of experimental examples:[Experimental example using human hair]
・MCT118 type test
[Experimental example using human nail]・Detection of mitochondrial DNA
・Detection of p53 gene
・MCT118 type test
[Experimental example using mouse body hair]・Detection of mitochondrial DNA
・Detection of p53 gene
[Experimental example using mouse nail]・Detection of mitochondrial DNA and p53 gene
etc.
Nippon Gene Co., Ltd. Website

http://www.nippongene.com

VII References
1）Wilson, M. R., Polanskey, D., Butler, J., Dizinno, J. A., Replogle, J. and Budowle, B.,
BioTechniques, 18 (4), 662-669 (1995)
2）Kasai, Kentaro, “Protein, Nucleic Acid, Enzyme”, 41 (5), 738-743 (1996)
3）Yoshii, T., Tamura, K., Ishiyama, I. “Journal of Japanese Society of Legal Medicine”,
46 (5), 313-316 (1992)
4）Higuchi, R., von Beroldingen, C. H., Sensabaugh, G. F. and Erlish, H. A., NATURE,
332 (7), 543-546 (1988)
5）Yoshii, T., Tamura, K., Taniguchi, T., Akiyama, K., and Ishiyama, I. “Journal ofJapanese Society of Legal Medicine”, 47 (4), 323-329 (1993)
6）Gill, P., Jeffreys, A. J. and Werrett, D. J., NATURE, 318 (12), 577-579 (1985)

We are grateful to Dr. Takanori Kobayashi, National Research Institute of Fisheries
Science, Fisheries Research Agency, for his kind instruction for the development of thisproduct.

- 12 -ISOHAIR Manual (ver.11)
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NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL
www.nippongene.com

ISOHAIR Manual (ver.11)
130930KH