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Генетический анализ. Лекция 8. Генетические задачи решаются легко только тогда, когда они предварительно уже решены другими. Поэтому необходимо предостеречь тех, кто впервые приступает к генетическому анализу, от уныния и пессимизма, если их первые попытки окажутся неудачными.

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Лекция 8

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8

8


8

, . , , , .


8

  • .

  • .


8


8

1983 (), .

( ) , (Newton, Graham, 1997).

Kary Baron Mullis

. () ( ), , (random primedamplification ); , () , - STS- (Sequence-TaggedSites).


8

. Random PCR

1990 , , . . (Williams et al., 1990) RAPD(Random Amplified Polymorphic DNA),

(Welsh, McClelland,1990) - AP-PCR (Arbitrary Primer PCR). (Caetano-Anolles et al., 1991) - DAF (DNA Amplification Fingerprinting)( ., 1992) - - ( ). , , .

:

"" , , .


8

Random PCR ( )


8

. Random PCR

, . - , , .

rPCR (Random Polymerase Chain Reaction ). ( , , ). , 3'- (Froussard, 1993).

RAPD (Random Amplified Polymorphic DNA ). RAPD- : , , , ; . RAPD- : o 50 80% G+C , 10 .


8

. Random PCR

RAPD- : RAPD- 5-12 ; RAPD- ; RAPD / ; RAPD -, .

AP-PCR (Arbitrarily Primed Polymerase Chain Reaction ). , (15- 25 ). , , ( "" ), "" . .

DAF (DNA Amplification Fingerprinting - ). DAF 5-8 . . DAF 3-10 , -, RAPD- . DAF - .


8

. Random PCR

  • RAPD. : ;

  • ;

  • ;

  • .

  • , :

  • "" () , , ;

  • , , ;

  • : -, .


8

Random PCR (RAPD, DAF, AP-PCR), AFLP

RAPD- Oligo 12( , )

RAPD, DAF, AP-PCR, AFLP. (RAPD, AP-PCR), (DAF, AFLP). : "1" - ( ) "" - ( ), . , ( ) , , . () (). , , ( ) . , , 670 .. Oligo 12 - Oligo 12670.


8

Random PCR (RAPD, DAF, AP-PCR), AFLP; CAP

RAPD- , , . . , , . , . . .

RAPD- . , .

RFLP, . ( ) .

RFLP- Hindlll Rbkl 12 ( , )

: 1 : 2220 .. ( ), 2 : 2120 .. 100.., 2 : 1660 .. 560 ..

Rbkl 12(HindIII):

1 , 2-5,7 , 6 , 8


8

. Random PCR

, RAPD- , - , , . , , RAPD- , , , , .. , . - , RAPD- , .. . , , . , .

. , . .


8

. STS PCR

, - STS (Sequence-TaggedSites). SCAR, EST, ISSR, CAPS, STMS ., . SCAR(Sequence-Characterised AmplifiedRegions), - , , , , (Antolin, 1996).

EST (Expressed Sequence Tag)- , , , , , , (Karp et al., 1997).

, , SSR- (SimpleSequence Repeats), STMS- (Sequence-Tagget Microsatellites), .


8

. STS PCR

- , , .

:

VNTR (Variable Number of Tandem Repeats - ). () 1-6 (, SSR - Simple SequenceRepeat) 9-10 (). , - . /, , . . . , .

5 . "-" . , , 5 0,01%.


8

. VNTR

- (, ) , . ( >1%)


8

. VNTR

- ( "" ). , , ( ) ( ) .

VNTR-


8

. STS PCR

SCAR (Sequence-Characterised Amplified Regions - ).

-, ). , EST- (ExpressedSequence Tag - ). SCAR- CAPS- (Cleaved Amplified PolymorphicSequence - - ), PCR-RFLP.

SCAR- ( )


8

(SNP)

SCAR- ( )


8

. STS PCR

. . , 100-. . ( 20-), . , , .

. , , . , , . - . , , , '- , . .


8

. STS PCR

. , , , (-, , 3'- ). , -, '- (dG)- . "" , 3'- (dC)- (dG-, "'" , 3'- .

STS-, . -.

.


8


8

. .

, : 1) , ( SYBR Green);

2) , PCR- ( TaqMan, Molecular Beacons LightCycler).


8

. SYBR Green

  • SYBR Green -SYBR Green(Morrison et al., 1998). SYBR Green , (- ) (). , , , .

  • : SYBR Green , .

  • :

  • SYBR Green . ;

  • ;

  • - ( , , TaqMan, ) , , .


8

. TaqMan

TaqMan 5'- (Held et al., 1996). 2 , , , - (), 5'- (), 3'- (). , . . . , , , - 5'- . , . , .


8

. TaqMan

, , , TaqMan, SYBR Green. , TaqMan , , .

, , - , .


8

. Molecular Beacon & Ligft Cicler

Molecular Beacons TaqMan , ( , , ) (Mhlanga, Malmberg, 2001). , " " (stem-loop), . , .

LightCycler , (Wittwer et al., 1997). , 3'- (), , 5'- (), , 1-3 . , . .


8

.

Real-Time qPCR. (, ), ().

SYBR Green , , . -. , , (-dl/dT), , . - , . . . .


8

.

() () QZag12 ( ) .

, : -, , . , 5-7 .

TaqMan , , . -, ( ) RealTime-.


8

.

TaqMan .

: 1 , 2 , 3 .

: , , , . ( ) , , .


8

.


8

.


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