1- 2

( ) [YMBAEI KAI YNTMHEI: O , 5' 3' , . O

DNA ,

. A ,

(5' 3') .

bp = base pair ( DNA) / nt = nucleotide ( . )

kb = kilobase = 1000 bp 1000 nt, .

ss = single stranded = / ds = double stranded = ]

O 1970 DNA . O DNA DNA ( ) in vitro . H , DNA . MEPIKE YIKOXHMIKE IIOTHTE TOY DNA H DNA . T :

A-T (2 ) G-C (3 )

DNA (ds) (ss). H DNA . O GC DNA, , .

1

1- 2 . A ss DNA, ss RNA ssDNA ssRNA ( RNA, DNA).

DNA ( DNA): AOMONH EIIKN AHOYXIN NOYKEKN OEN

1. M (buoyant density) ( DNA). E DNA = . O GC , .

M CsCl , DNA , . , , DNA . E , DNA , . M , , DNA. M , .

2. E DNA, .. DNA. O DNA , ( ). T DNA () (), .

3. DNA , ( ) , . T - DNA

2

1- 2 (DNase) DNA - DNase DNA.

T DNA RNA. T DNA RNA , DNA, .

ENZYMA EPIOPIMOY

E dsDNA . O (.. EcoRI Escherichia coli, Sau3A Staphylococcus aureus) : 4 6 bp (.. CTGCAG

GGCC). :

, (blunt ends). A DNA (staggered cuts) , 2 4 . T 5' 3' , . E , (sticky ends).

M ( ) DNA, ( DNA). DNA 6bp 46 bp = 4096 bp, 4bp , 44 bp = 256 bp.

XAPTE EPIOPIMOY: T DNA, ( ) DNA. , DNA

3

1- 2 ( ). M ( ) . T : - DNA (Southern blots):

A B. - DNA

( ).

- - .

, DNA ( , 5-50 x 103 bp), . H () .

E 200 () - 106 bp, ( ), 50 bp. E in vitro , . O DNA, DNA.

KNOOIHH DNA

DNA, , . T dsDNA (nicks), . H 4 , , . () . A DNA , (lagging) DNA. T

4

1- 2 dsDNA A .

DNA ( ) DNA : DNAs (cloning vectors). A .. A, DNA A. X DNA DNA (. 1).

E . ,

5

1- 2 (insert) DNA . E DNA ' .

K A (.. ). T E. coli, DNA. E , . H .

E mRNA . A cDNA (copy DNA), DNA mRNA . T cDNA mRNA (reverse transcriptase), DNA RNA. E cDNA , . 3' (.. CCC...CC) cDNA ( GGG...GG). A (terminal transferase). H .

OPEI KNOOIHH: T DNA : 1. I DNA ( - origin of

replication) E. coli. 2. E (.. ,

). 3. E . E DNA.

T ( 50) . () (, , ),

6

1- 2 . H ( ) DNA CsCl .

O M13. K DNA , DNA . H ( ) . O ~50 kb, 20 kb E. coli DNA. H . T M13 DNA DNA , (. ).

M . ..: - . - E. coli. - E. coli , ..

. -

-.

TPATHIKE KNOOIHH cDNA . , . H 17 kb ( ) (..) 17 / 170000 = 10-4 ( 170000 kb). E, 99% 10 , . 100000 . H , , . M :

(1) Y .

7

1- 2 : (.. ) - cDNA , ( , . cDNA )- ( ) ( ) - RNA ( cDNA) - .. ..

(2) T , . A () - . E "" .

(3) E , . (E. coli). O in situ (cDNA) (. ). E / ( ).

POIOPIMO AHOYXIA TOY DNA (SEQUENCING)

DNA. H (Maxam and Gilbert) .

H (Sanger) DNA, in vitro. H DNA E. coli (dATP, dTTP, dGTP, dCTP, ) DNA . T ' 2',3'- (ddATP, ddGTP .). H ddNMP 3'

8

1- 2 , 3' -OH . O .. ddATP dATP A. E , A, G, C T, ddNTP . M DNA.

9

1- 2

ONIIAKH OMH H DNA () ' . DNA. , . .

POKAPYTIKA ONIIA H DNA mRNA . T mRNA , . . ( mRNA) RNA . , , (reading frame) . H 3' mRNA (3' untranslated region or 3'UTR) cis- . H 5' flanking region (5' ) cis- .

10

1- 2

11

EYKAPYTIKA ONIIA T mRNA : cap 5' ( ) 3'. T DNA mRNA . T mRNA , , , RNA . H (splicing) cDNA . E , . T 5' 3' flanking . 5' UTR 3 UTR , 3' UTR RNA.

H :

GENE 1 5 FLANKING GENE 2 GENE 3

3 UTR 5 UTR

mRNA

EXON 3 EXON 15 FLANKING EXON 2

5 UTR 3 UTR

INTRON 2INTRON 1

1 32mG-PPP AAAAAAAAAAAAAAAAAA

O mRNA