Non-radioactive detection of dot-blotted genomic DNA for species identification

HAL Id: hal-00893906

https://hal.archives-ouvertes.fr/hal-00893906

Submitted on 1 Jan 1991

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Non-radioactive detection of dot-blotted genomic DNA

for species identification

Tr Chen, C Dorotinsky

To cite this version:

Non-radioactive detection of dot-blotted

genomic

DNA

for

_species

identification

TR

Chen, C

_Dorotinsky

American

Type

Culture

Collection,

12301 Parklawn Drive,

Rockville,

MD

_20852,

17SA

(Proceedings

of the 9th

European Colloquium

on

_Cytogenetics

of Domestic

Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

species identification

_/

_{biotinylation}

_/

_genomic DNA

INTRODUCTION

The _purpose of this

study

was to find a

_simple

DNA-based

_procedure

for

_species

identification to be used for

quality

control in cell culture

_systems.

_{Isoenzymology}

and

_cytogenetics

are

_frequently

used for

_identifying

species origin

of a cell line.

However,

these methods are labor intensive and

technically

demanding

and results are sometimes indefinite. Additional

_simple,

sensitive and

_reproducible

methods

to

_identify

_species

would be a welcome progress.

_Herein,

we

_report

a

procedure

to

_identify

the

_genomic

DNA

_(gDNA)

of an unknown

_species,

dot-blotted on a

nitrocellulose

filter,

by hybridizing

with

biotinylated,

non-radioactive DNA

probes

from a known

species. By

selecting

the

appropriate probes,

this

procedure

can be

used to

_distinguish

_species

_{gDNAs clearly.}

MATERIALS AND METHODS

Materials

Cell lines and

_plasmid

DNAs

_containing

different chromosome

_segments

used for this

_study

are listed in tables I and II.

They

are all

_deposited

at the ATCC.

Procedures

DNA extraction. The standard

_{phenol-chloroform}

method was used for DNA

extraction and

_purification

_(Maniatis

et

_al,

_1982).

The amount of DNA was

measured

_by

spectrophotometry.

Biotinylation.

Both biotin-14-dATP

_(BRL)

and biotin-21-dUTP

_(BioRad)

were

used. Methods to

_biotinylate

DNA

_probes

followed manufacturers’ recommenda-tions with

_slight

modifications as follows:

1)

1.5-2.0 _pg of the DNA

probe

was used

per

reaction;

2)

reaction time was extended to 5-7 h before

_{being stopped}

with 0.3 M

_{ethylenediaminetetraacetic}

acid

_(EDTA);

and

₃₎

the reaction solution was

Blotting. Following

the standard method

_(Maniatis

et

_al,

_1982),

denatured

_gDNAs

were blotted onto the nitrocellulose membrane filters

_using

a minifold

apparatus

(Schleicher

&

_Schuell,

_Inc).

The blotted membrane was incubated in the vacuum oven for 2-3 h at 80°C.

Hybridization.

15-30

_al

of

_biotinylated

_probe

were used for each reaction that

used a 70

CM

2

filter and 3.5 ml of

_{prehybridization}

or

_{hybridization}

solution

supplemented

with an

_appropriate

amount of

_freshly

denatured salmon _sperm DNA

_(Maniatis

et

_al,

_1982).

_{Prehybridization}

and

_{hybridization}

incubations were,

respectively,

3 h and

_overnight

at 42°C.

Detection.

_Biotinylated

DNA was detected with

_{streptoavidin-alkaline}

phos-phatase conjugate

according

to the methods recommended

_by

the manufacturer.

RESULTS

Qualitative

and

_quantitative

differences in

_{hybridization}

to the

_gDNA

on the

ni-trocellulose membranes were

_clearly

demonstrated with non-radioactive

biotiny-lated DNA

_probes.

Positive

_{hybridization}

was revealed

_by

the

_{streptoavidin-alkaline}

phosphatase

conjugate

detection

system.

When monitored

_properly,

_{50 ng of}

_gDNA

produced,

within 5

_min,

the distinct

_deep

blue color of the maximum +5 scale

adopted

for our evaluation. This method is

_{simple, reproducible}

and

uncompromis-ing.

Tables I and II summarize

_{hybridization}

results. Probes

_using

_gDNA

from human

(Hsa),

mouse

_(Mmu)

and rat

_(Rno)

cell lines showed a lack of

_{hybridization}

between

primates

and rodents

_{(fig 1).}

_By

contrast,

weak to moderate cross-reactions were

observed _among three rodent

_{species, Mmu,}

Rno and Chinese

_(Ch)

hamster

_(Cgr)

(fig

lb,

c).

Thus,

gDNA

can be used as the DNA

_probe

to

_{distinguish distantly}

Within

_primates,

human

_gDNA

showed substantial

_{cross-hybridization}

with

gDNAs

from

_chimpanzee

_(Ptr),

_gorilla

_(Ggo),

_orangutan

_(Ppy),

_{African green}

mon-key

(Cae)

and marmoset

_(Sni)

_{(eg, fig la).}

When DNAs from human chromosome N8 or an N13

segment

were used as the

_{probe, they}

cross-reacted with all

_primate

species

studied here

_(fig

_2a,

_b),

_producing

a result similar to that of the

_gDNA.

These data demonstrate the substantial

_homology

in

these

species.

Alphoid

satellite DNAs

₍

_S

_DNA)

from human chromosomes N10 and N12

dis-tinguished

African _green

_monkey

and marmoset from the

_remaining

4

_species

(fig

2c,

d).

The

_non-alphoid

sDNA from human N22

_suggested

a difference between

chimpanzee

and the

_Hsa-Ggo-Ppy

_species

_{(fig 2e).}

_Alphoid

sDNA from human N1 reacted

_similarly

to N10 and

N12,

except

that with the

_orangutan

_gDNA

which

ap-peared

relatively

weak

_{(fig 2f).}

Alphoid

sDNA from N8 and

N18, however, clearly

distinguished

not

_only

the Cae-Sni from the other 4

_species,

but also the

_Hsa-Ppy

from the

_Ptr-Ggo

_(fig

_2g,

_h).

Experiments

to further discriminate between these

probes

for

_species

identification are

_currently

in progress.

Probe DNA from an

_N13q

_segment

identified the man-mouse cell

hybrid

H99

but not the

_hybrid

H101

_{line, which,}

_{respectively, possessed only}

human

_t(13q17q)

or N17

_{(fig 3).}

The

potential

use of this

procedure

for

syntenic

gene-chromosome

mapping

by

means of somatic cell

hybridization

is

proposed.

DISCUSSION AND CONCLUSION

Since the isolation

_{of genomic}

DNA is a

simple,

routine

procedure,

and the

_reagents

for

_{biotinylation}

and detection are available

_commercially

in kit

_form,

this

non-radioactive

_{biotinylation procedure}

can be

_performed

_by

most laboratories. No

cross-hybridization

was detected

_using

rodent or human

_gDNAs.

Mouse and rat

gDl!TAs

readily

distinguish

distantly

related

species.

Since most cell lines are derived

from these 4

_species,

this

_procedure

is a useful means for

quality

control of cell cultures.

_{Moreover, plasmid}

DNA

_containing

a chromosome

_segment

can be used to

_identify

the presence of that chromosome in a

_hybrid

cell line genome; it

provides

a

_simple

alternative for the identification of chromosomes and is also useful for gene

mapping

studies

_by

somatic cell

hybridization.

Specific

DNA

_probes

can be used to

_{identify phylogenetically}

close

_species.

DNA from some human chromosomes or chromosome

_segments

cross-reacted with 6

primate species.

However,

alphoid

DNAs from different human chromosomes are more

specific,

since

_they

show differential cross-reactions. The molecular

_aspects

of

the

_complexity

of these

_alphoid

sDNAs have been the

subject

of many studies

(see

reviews

_by

_Singer,

_1982;

Willard and

_Waye,

_1987).

_{Cytogenetically,}

this

complexity

is also

_apparent,

since individual chromosome identification

_requires

a

_step

for

chromosome in situ

_suppression

_(CISS)

to further eliminate the

_non-specific

oligo-DNA _repeats

_(Lichter

et

_al,

_1990).

As also demonstrated

_herein,

_alphoid

SDNA

probes

from different human chromosomes

_recognized

_{species-specific properties}

among the 6

primate species

tested.

Thus,

this DNA

_subfamily

can be used not

only

to

_identify

individual chromosomes

_(Pinkel

et

al, 1986;

Lichter et

al, 1988,

1990),

but also to

_distinguish

between

_closely

related

_species.

_Moreover,

_{it may}

give

inferences as to the

_phylogenetic

_relationship

between

_species,

as the data on

N8 and N18

alphoid

DNAs

_suggest

a closer

_affinity

between human and

orangutan

than between human and

_{chimpanzee-gorilla.}

REFERENCES

Lichter P,

Chang-Tang CJ,

Call

_K,

Hermanson

_G,

Evans

_GA,

Housman

_D,

Ward DC

(1990)

High-resolution mapping

of human chromosome 11

_by

in situ

_{hybridization}

with cosmid clones. Science

247,

64-69

Maniatis

_T,

Fritsch

_EF,

Sambrook J

₍₁₉₈₂₎

Molecular

_Cloning:

A

_Laboratory

Manual. Cold

_Spring

Harbor

_Laboratory,

Cold

_{Spring Harbor,}

NY

Pinkel

D,

Straume

T,

Gray

JW

₍₁₉₈₆₎

_{Cytogenetic analysis using quantitative, high}

sensitivity,

fluorescence

_{hybridization.}

Proc Natl Acad Sci USA 83, 2934-2938

Singer

MF

₍₁₉₈₂₎

_{Highly repeated}

_{sequences in} mammalian genomes. Iret Rev

Cytol76,

67-112