Y CHROMOSOME SINGLE NUCLEOTIDE POLYMORPHISMS TYPING BY SNaPshot MINISEQUENCING
Noveski P, Trivodalieva S, Efremov GD, Plaseska-Karanfilska D
*Corresponding Author: Dr. Dijana Plaseska-Karanfilska, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Av. Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel.: +389-2-3235-410; Fax: +389-2-3115-434; E-mail: dijana@manu.edu.mk
page: 11

MATERIALS AND METHODS

Materials. We have studied 343 DNA male DNA samples from the DNA bank at our institution, of which 211 were Macedonians, 111 Albanians and 21 Roma, Serbs or Turks. The study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia (R. Macedonia).

Methods. The set of 28 Y-SNP markers was grouped hierarchically into five multiplex polymerase chain reaction (mPCR)/primer extension reactions, so as to determine the most frequent haplogroups using one or two multiplexes (Figure 1). Most of the PCR and minisequencing primers have already been described [8], the remainder were designed by us.

Multiplex Polymerase Chain Reaction. The PCR primers were designed to give a variety of PCR fragment sizes, that allow their separation by polyacrylamide gel electrophoresis (Table 1 and Figure 2). The PCR multiplexes were performed in 25 L final volume, with 1X Reaction buffer, 300 M of dNTPs, 2 mM of MgCl2, 2U of AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA, USA) and 10 ng of genomic DNA. Cycling conditions were 10 min. at 95C, then 35 cycles of 1 min. at 95C, 1 min. 15 s. at 58C, 3 min. at 72C and a final extension at 65°C for 15 min.

Multiplex Minisequencing. Before a single base extension (SBE), 1 L of PCR product was cleaned up with 0.5 L of ExoSAP-IT (USB Corporation, Clevelend, OH, USA) and incubated at 37C for 60 min. followed by 15 min. at 85C to inactivate the enzyme.

Multiplex single base extension reactions were performed in a 5 L final volume, combining 2 L of SNaPshot ready reaction mix (Applied Biosystems), 1.5 L of cleaned PCR product and 1.5 L extension primers. The minisequencing primers were 5' tailed with a poly-C sequence to produce extension products 21 to 51 nucleotides long to allow separation by capillary electrophoresis (Table 2). The cycling conditions were 10 s. at 96C, 10 s. at 50°C and 30 s. at 60C, for 25 cycles.

To remove unincorporated ddNTPs, the final products were incubated with 1U of shrimp alkaline phosphatase (USB Corporation) for 1 hour at 37C, and 15 min. at 85C to inactivate the enzyme.

Capillary Electrophoresis. The SNaPshot (Applied Biosystems) products were separated by capillary electrophoreis on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). Analysis of electropherograms was performed using the GeneScan 3.1 software (Applied Biosystems) and the size of fragments was determined on the basis of GeneScan-120 LIZ size standard (Applied Biosystems) (Figure 3).





Number 26
VOL. 26(1), 2023
Number 25
VOL. 25(2), 2022
Number 25
VOL. 25 (1), 2022
Number 24
VOL. 24(2), 2021
Number 24
VOL. 24(1), 2021
Number 23
VOL. 23(2), 2020
Number 22
VOL. 22(2), 2019
Number 22
VOL. 22(1), 2019
Number 22
VOL. 22, 2019 Supplement
Number 21
VOL. 21(2), 2018
Number 21
VOL. 21 (1), 2018
Number 21
VOL. 21, 2018 Supplement
Number 20
VOL. 20 (2), 2017
Number 20
VOL. 20 (1), 2017
Number 19
VOL. 19 (2), 2016
Number 19
VOL. 19 (1), 2016
Number 18
VOL. 18 (2), 2015
Number 18
VOL. 18 (1), 2015
Number 17
VOL. 17 (2), 2014
Number 17
VOL. 17 (1), 2014
Number 16
VOL. 16 (2), 2013
Number 16
VOL. 16 (1), 2013
Number 15
VOL. 15 (2), 2012
Number 15
VOL. 15, 2012 Supplement
Number 15
Vol. 15 (1), 2012
Number 14
14 - Vol. 14 (2), 2011
Number 14
The 9th Balkan Congress of Medical Genetics
Number 14
14 - Vol. 14 (1), 2011
Number 13
Vol. 13 (2), 2010
Number 13
Vol.13 (1), 2010
Number 12
Vol.12 (2), 2009
Number 12
Vol.12 (1), 2009
Number 11
Vol.11 (2),2008
Number 11
Vol.11 (1),2008
Number 10
Vol.10 (2), 2007
Number 10
10 (1),2007
Number 9
1&2, 2006
Number 9
3&4, 2006
Number 8
1&2, 2005
Number 8
3&4, 2004
Number 7
1&2, 2004
Number 6
3&4, 2003
Number 6
1&2, 2003
Number 5
3&4, 2002
Number 5
1&2, 2002
Number 4
Vol.3 (4), 2000
Number 4
Vol.2 (4), 1999
Number 4
Vol.1 (4), 1998
Number 4
3&4, 2001
Number 4
1&2, 2001
Number 3
Vol.3 (3), 2000
Number 3
Vol.2 (3), 1999
Number 3
Vol.1 (3), 1998
Number 2
Vol.3(2), 2000
Number 2
Vol.1 (2), 1998
Number 2
Vol.2 (2), 1999
Number 1
Vol.3 (1), 2000
Number 1
Vol.2 (1), 1999
Number 1
Vol.1 (1), 1998

 

 


 About the journal ::: Editorial ::: Subscription ::: Information for authors ::: Contact
 Copyright © Balkan Journal of Medical Genetics 2006