chapter 4 molecular cloning methods
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Chapter 4Molecular Cloning Methods
Jay D. Hunt, Ph.D.Department of Biochemistry and Molecular Biology
CSRB 4D1568-4734
jhunt@lsuhsc.edu
• 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
• 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
GAATTCCTTAAG
GAATTCCTTAAG
Cloning
GCTTAA
AATTCG
Digest with EcoRI
GCTTAAAATTC
G
Hybridize
GAATTCCTTAAG
Ligation
-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
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
Digest with BamHI Partial Digest with Sau3A
Isolate pieces 12-20 KB in lengthCombine
Package into phage heads
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
Denaturation (94°C)
+ +
Annealing (37-65°C)
Extension (72°C)
Template Primers dNTPs
First round complete
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 cloningVI. Labeling DNA with nick translation
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCR
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCRVIII.5’ RACE
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
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