Chapter 4Molecular Cloning Methods

Jay D. Hunt, Ph.D.Department of Biochemistry and Molecular Biology

CSRB 4D1568-4734

jhunt@lsuhsc.edu

I. Restriction Endonucleases

• Restriction endonucleases– Restriction - Bacterial encoded restriction

endonucleases restrict bacteriophages to only one host strain.

– Endonuclease - Restriction endonucleases cleave nucleic acids in the middle.

• Subclasses of restriction endonucleases:– Type I - Recognize specific sequences and cleave DNA

at a nonspecific site > than 1,000 bp away– Type II - Recognize palindromic sequences and cleave

within the palindrome– Type III - Recognize specific 5-7 bp sequences and

cleave 24-27 bp down stream of the site.

• Type II restriction endonucleases are the most useful class, as they recognize specific palindromic sequences in DNA and cleave the phospodiester bonds in the ribose backbone within the palindrome

• A palindrome is anything that reads the same forwards and backwards:– Mom– Dad– Tarzan raised Desi Arnaz rat.– Able was I ere I saw Elba– Doc note I dissent, a fast never prevents a fatness; I diet on

cod.– Do good? I? No! Evil anon I deliver. I maim nine more hero-

men in Saginaw, sanitary sword a-tuck, Carol, I–lo–rack, cut a drowsy rat in Aswan. I gas nine more hero-men in Miami. Reviled, I (Nona) live on. I do, O God!

• In DNA, palindromes are defined as double stranded DNA that reads the same 5’ to 3’

• The EcoRI cutting site:– 5'-GAATTC-3'– 3'-CTTAAG-5'

• The HindIII cutting site:– 5'-AAGCTT-3'– 3'-TTCGAA-5'

Types of recognition sites:4 bp6 bp8 bp

44 = 256 bp46 = 4,096 bp48 = 65,536 bp

Table 4.1

Figure 4.1

• Type II restriction endonucleases cut only at specific palindromic sites; therefore, “sticky ends” result from DNA cleavage. Fragments of DNA cut with the same enzyme will hybridize to these sticky ends.

Always indicate 5’ and 3’ ends of BOTH strands.Always indicate 5’ and 3’ ends of BOTH strands.

3'CTTAAG5' 3'CTTAA5' 3'G5'

5'GGATCC3'3'CCTAGG5'

5'G3' 5'GATCC3'3'CCTAG5 3'G5'

Eco RI

Bam HI

5'GAATTC3' 5'G3' 5'AATTC3'

Hin dIII 5'AAGCTT3'3'TTCGAA5'

5'A3' 5'AGCTT3'3'TTCGA5' 3'A5' 5’ overhang

5’ overhang

5’ overhang

5'GATATC3'3'CTATAG5'

5'GAT3' 5'ATC3' 3'CTA5' 3'TAG5'EcoRV Blunt end

3'GACGTC5' 3'G5' 3'ACGTC5'5'CTGCAG3' 5'CTGCA3' 5'G3'Pst I 3’ overhang

I. Restriction EndonucleasesII. Cloning

GAATTCCTTAAG

GAATTCCTTAAG

Cloning

GCTTAA

AATTCG

Digest with EcoRI

GCTTAAAATTC

G

Hybridize

GAATTCCTTAAG

Ligation

Text Art Page 62

Figure 4.2

Figure 4.3

Origin of replication

At least one uniquerestriction site

A selectable marker

Figure 4.4

Figure 4.5

Figure 4.6

Multicloning site-peptide of -galactosidase

DNA fragment up to 5 KBcan insert

orip

-peptide of -galactosidase is encoded by lacZNH2-terminal portion

lacZ is disrupted by insert

-peptide is carried in genetically modified bacterialstrains. COOH-terminal portion

-complementation occurs.5-bromo-4-chloro-3-indolyl--D-Galactopyranoside (X-gal) ismetabolized resulting in bluecolonies

No -complementationoccurs. White colonies

Figure 4.7b

Addition of ligasewould causethis to seal

Without phosphategroup, ligationcannot occur

Phosphates are donatedby the insert

Ligation occurs

Figure 4.7a

Note that the phosphategroup is required forligation to occur.

Eco

RI

Eco

RI

Kpn

I

pUC18

lacZ

MCS

Sst I

EcoR

I

Kpn

I

Sma

I/Xma

I

Bam

HI

Xba

I

Sal I/Acc

I Hinc

II

Pst ISph

I

Hind

III

Eco

RI

Eco

RI

Kpn

I

Sst I

EcoR

I

Kpn

I

Sma

I/Xma

I

Bam

HI

Xba

I

Sal I/Acc

I Hinc

II

Pst I

Sph

I

Hind

III

5'-G AATTC-3'

3'-CTTAA G-5'Digestion with EcoRI

5'-G C-3'

3'-CTTAA CATGG-5'Digestion with EcoRI & Kpn I

Eco

RI

Eco

RI

Kpn

I

Sst I

EcoR

I

Kpn

I

Sma

I/Xma

I

Bam

HI

Xba

I

Sal I/Acc

I Hinc

II

Pst I

Sph

I

Hind

III

Digest both insert and vector with EcoRI and Kpn I

EcoR

I

Kpn

I

Figure 4.8

Required forlysogeniclifecycle

Required forlytic lifecycle(progenyproduced)

12 to 20 KB inserts

Genomic Library Construction

cos sitesBam

HI

Bam

HI

12-20 KB insert

Bam

HI

Bam

HI

Bam

HI

Bam

HI

Bam

HI

Bam

HI

Bam

HI

Bam

HI

Bam

HI

~4 KB

Too short, not viable

Sau3A, ~250 bp

-GGATCC--CCTAGG-

BamHI Sau3A

-GATC--CATG-

-G-CCTAG

GATC- -

-GGATC--CCTAG-

Digest with BamHI Partial Digest with Sau3A

Isolate pieces 12-20 KB in lengthCombine

Package into phage heads

Figure 4.9

DNA hybridization

Figure 4.10

Figure 4.11

40 to 50 KB inserts

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clones

GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTGCCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC

GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTG

CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC

Melt

GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTG

CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC

Probe

GCCGATTCCAGCTAGTCAAGG

CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC

CCACCGTACAAATAAGTTCAATCAGGGAACATGAC

GCCGATTCCAGCTAGTCAAGG

GCCGATTCCAGCTAGTCAAGG

Low stringency hybridization

Low stringency washing conditionsHigh salt concentration (0.3 M NaCl)Low temperature (20 to 30°C)Low organic solvent concentrations

CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC

CCACCGTACAAATAAGTTCAATCAGGGAACATGAC

GCCGATTCCAGCTAGTCAAGG

GCCGATTCCAGCTAGTCAAGG

Low stringency hybridization

High stringency washing conditionsLow salt concentration (0.03 M NaCl)High temperature (65°C)High organic solvent concentrations

GCCGAT

TCCAGC

TAGTCA

AGG

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCR

Figure 4.12

Denaturation (94°C)

+ +

Annealing (37-65°C)

Extension (72°C)

Template Primers dNTPs

First round complete

94°C

37-65°C

72°C

Second round complete

1

2

4

8

16

32

64

30 rounds of PCR =

1,073,741,824 (1.07 X 109) copies

40 rounds of PCR =

1,099,511,628,000 (1.1 X 1012) copies

Exponential Increase in Target DNA

From 1 copy of template DNA

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloning

Figure 4.13

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translation

Figure 4.14

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCR

Figure 4.15

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCRVIII.5’ RACE

Figure 4.16

I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCRVIII.5’ RACEIX. Expression vectors

Figure 4.17

Figure 4.19a

Figure 4.19b

Figure 4.20

Figure 4.21

Figure 4.22