(1) Amplification of human genomic DNA

Template:

Human genomic DNA
500 ng/50 PCR

Target regions:
-globin cluster and TPA gene

Applied volume:

5

0.4% SeaKem Gold Agarose (BMA)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(2) Amplification of human genomic DNA directly from human blood 

 

TaKaRa LA Taq is applicable to GC rich template by using specially designed buffers

(2x GC Buffer I, II). Performance was compared between LA PCR Kit Ver. 2.1 and GC

kit from Company A when the GC rich fragments containing CAG repeat were amplified.

 

 

 Template:
Human genomic DNA 100 ng/50 PCR Target:
Huntington's disease gene
(HD gene IT15 CAG repeat) Amplified size:
262 bp

(GC content: 73%)
358 bp

(GC content: 71.5%)

Applied volume:
8 /50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 (3) Amplification of 21.5 kbp human genomic DNA

 

Amplification efficiency of TaKaRa LA Taq was compared among various amount of

template when human genomic DNA was used as template. The result shows that

TaKaRa LA Taq can amplify even with lower amount of template.

 

 

 Amplified size:
21.5 kbp

Applied volume:
8 /50

1% Agarose L03  "TAKARA"

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 (4) Amplification of 5'-region of mRNA using 5'-Full RACE Core Set

 

Takara'S 5'-Full RACE Core Set (Cat.# 6122) achieves highly efficient amplification of

an unknown 5'-end region of mRNA in conjunction with TaKaRa Ex Taq, TaKaRa LA

Taq, or LA PCR Kit Ver. 2.1.

The 5'-region of human -actin mRNA was amplified through 5'-RACE method using of

poly(A)+ RNA of HeLa Cell.

The sequence of the amplified fragment was analyzed after subcloned by TA cloning.

The fragment was confirmed to contain the sequence to -actin mRNA CAP site as reported.

 

     

 

 

 

 

 

 

 

Schematic diagram of 5'-RACE using Takara's

5'-Full RACE Core Set

(1) Synthesize 1st strand cDNA by reverse transcription from

      target mRNA using 5'-end-phosphorylated RT Primer

      which is specific to the target RNA

(2) Degradation of hybrid DNA-RNA to separate RNA by

     treatment with RNase H

(3) Circularization of single-stranded cDNA or formation of      concatemers by RNA Ligase

(4) DNA Amplification by PCR

 

References

1)  Frohman, M. A., Dush, M. K., Martin, G. R. (1988)

     Proc. Natl. Acad. Sci. USA, 85, 8998-9002.

2)  Maruyama, I. N., Rakow, T. L., Maruyama, H. I.

     (1995) Nucleic Acid Research, 23, 3796-3797.