chapter 4 molecular cloning methods jay d. hunt, ph.d. department of biochemistry and molecular...
TRANSCRIPT
![Page 1: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/1.jpg)
Chapter 4Molecular Cloning Methods
Jay D. Hunt, Ph.D.Department of Biochemistry and Molecular Biology
CSRB 4D1568-4734
![Page 2: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/2.jpg)
I. Restriction Endonucleases
![Page 3: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/3.jpg)
• Restriction endonucleases– Restriction - Bacterial encoded restriction
endonucleases restrict bacteriophages to only one host strain.
– Endonuclease - Restriction endonucleases cleave nucleic acids in the middle.
![Page 4: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/4.jpg)
• 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
![Page 5: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/5.jpg)
• 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!
![Page 6: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/6.jpg)
• 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
![Page 7: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/7.jpg)
Table 4.1
![Page 8: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/8.jpg)
Figure 4.1
![Page 9: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/9.jpg)
• 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.
![Page 10: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/10.jpg)
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
![Page 11: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/11.jpg)
I. Restriction EndonucleasesII. Cloning
![Page 12: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/12.jpg)
GAATTCCTTAAG
GAATTCCTTAAG
Cloning
GCTTAA
AATTCG
Digest with EcoRI
GCTTAAAATTC
G
Hybridize
GAATTCCTTAAG
Ligation
![Page 13: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/13.jpg)
![Page 14: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/14.jpg)
Text Art Page 62
![Page 15: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/15.jpg)
![Page 16: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/16.jpg)
Figure 4.2
![Page 17: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/17.jpg)
Figure 4.3
Origin of replication
At least one uniquerestriction site
A selectable marker
![Page 18: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/18.jpg)
Figure 4.4
![Page 19: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/19.jpg)
Figure 4.5
![Page 20: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/20.jpg)
Figure 4.6
Multicloning site-peptide of -galactosidase
![Page 21: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/21.jpg)
DNA fragment up to 5 KBcan insert
orip
![Page 22: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/22.jpg)
-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
![Page 23: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/23.jpg)
Figure 4.7b
Addition of ligasewould causethis to seal
Without phosphategroup, ligationcannot occur
Phosphates are donatedby the insert
Ligation occurs
![Page 24: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/24.jpg)
Figure 4.7a
Note that the phosphategroup is required forligation to occur.
![Page 25: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/25.jpg)
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
![Page 26: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/26.jpg)
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
![Page 27: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/27.jpg)
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
![Page 28: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/28.jpg)
Figure 4.8
Required forlysogeniclifecycle
Required forlytic lifecycle(progenyproduced)
12 to 20 KB inserts
![Page 29: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/29.jpg)
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
![Page 30: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/30.jpg)
Sau3A, ~250 bp
-GGATCC--CCTAGG-
BamHI Sau3A
-GATC--CATG-
-G-CCTAG
GATC- -
-GGATC--CCTAG-
![Page 31: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/31.jpg)
Digest with BamHI Partial Digest with Sau3A
Isolate pieces 12-20 KB in lengthCombine
Package into phage heads
![Page 32: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/32.jpg)
Figure 4.9
![Page 33: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/33.jpg)
DNA hybridization
![Page 34: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/34.jpg)
Figure 4.10
![Page 35: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/35.jpg)
Figure 4.11
40 to 50 KB inserts
![Page 36: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/36.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clones
![Page 37: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/37.jpg)
GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTGCCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC
GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTG
CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC
Melt
GGTGGCATGCCGATTCCAGCTAGTCAACCGTACTG
CCACCGTACGGCTAAGGTCGATCAGTTGGCATGAC
Probe
GCCGATTCCAGCTAGTCAAGG
![Page 38: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/38.jpg)
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
![Page 39: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/39.jpg)
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
![Page 40: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/40.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCR
![Page 41: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/41.jpg)
Figure 4.12
![Page 42: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/42.jpg)
Denaturation (94°C)
+ +
Annealing (37-65°C)
Extension (72°C)
Template Primers dNTPs
First round complete
![Page 43: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/43.jpg)
94°C
37-65°C
72°C
Second round complete
![Page 44: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/44.jpg)
1
2
4
8
16
32
64
![Page 45: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/45.jpg)
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
![Page 46: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/46.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloning
![Page 47: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/47.jpg)
Figure 4.13
![Page 48: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/48.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translation
![Page 49: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/49.jpg)
Figure 4.14
![Page 50: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/50.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCR
![Page 51: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/51.jpg)
Figure 4.15
![Page 52: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/52.jpg)
I. Restriction EndonucleasesII. CloningIII. Probes to detect specific clonesIV. PCRV. cDNA cloningVI. Labeling DNA with nick translationVII.Cloning with Reverse Transcriptase-PCRVIII.5’ RACE
![Page 53: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/53.jpg)
Figure 4.16
![Page 54: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/54.jpg)
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
![Page 55: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/55.jpg)
Figure 4.17
![Page 56: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/56.jpg)
Figure 4.19a
![Page 57: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/57.jpg)
Figure 4.19b
![Page 58: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/58.jpg)
Figure 4.20
![Page 59: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/59.jpg)
Figure 4.21
![Page 60: Chapter 4 Molecular Cloning Methods Jay D. Hunt, Ph.D. Department of Biochemistry and Molecular Biology CSRB 4D1 568-4734 jhunt@lsuhsc.edu](https://reader036.vdocuments.us/reader036/viewer/2022062519/5697bfb51a28abf838c9da98/html5/thumbnails/60.jpg)
Figure 4.22