1. Recombinant DNA
Chapter 18

2. Learning Objectives Define Clone and DNA Cloning
List the three steps of production of recombinant DNA
Describe the characteristics and uses of a restriction endonuclease
Diagram the process of identifying a transformed bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

3. Learning Objectives Explain the uses of RFLPs
Describe the process of producing a transgenic organism, and explain its usefulness

4. DNA cloning
Clone: genetically identical cells or individuals derived from a single ancestor
DNA cloning: a method of producing a large amount of the DNA of interest
Large amounts of identical pieces of DNA enable us to manipulate and recombine genetic material

5. DNA Technologies
DNA technologies are used in molecular testing for many human genetic diseases
DNA fingerprinting used to identify human individuals and individuals of other species
Genetic engineering uses DNA technologies to alter the genes of a cell or organism
DNA technologies and genetic engineering are a subject of public concern

6. Recombinant DNA DNA from two or more sources joined together
DNA of interest can be spliced into bacterial plasmids (recombination)
Plasmids replicate (amplification)
Plasmids (DNA) are extracted (isolation)

7. Endonucleases
Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites
Restriction fragments result
Sticky ends have unpaired bases at cuts which will hydrogen bond
Ligase stitches together paired sticky ends

8. Restriction site for EcoRI
DNA
EcoRI restriction enzyme cleaves sugar–phosphate
backbones at arrows. 1
Sticky end
Sticky end
DNA fragments with the same sticky ends can pair. Shown here is a DNA fragment inserting between two other DNA fragments, as happens when inserting a DNA fragment into a bacterial plasmid. 2
Another DNA fragment
produced by EcoRI digestion
Figure 18.3
The restriction site for the restriction enzyme EcoRI, and the generation of a recombinant DNA molecule by complementary base pairing of DNA fragments produced by digestion with the same restriction enzyme.
Nick in sugar–phosphate backbone
Nicks in sugar–
phosphate backbones
are sealed by DNA ligase. 3
Recombinant DNA molecule
Fig. 18-3, p. 374

9. Recombinant DNA Restriction endonucleases
Each type is specific for a four to eight base pair long palindromic recognition sequence of DNA
Palindrome- reads the same on each strand 3’ to 5’ like GAATTC
CTTAAG

10. DNA fragments with sticky ends
Gene of
interest
Restriction
site
lacZ+
gene
Cell
ampR
gene
Plasmid
cloning
vector
DNA fragments with sticky ends
Cut plasmid cloning vectors with a restriction
enzyme to produce sticky ends
Figure 18.4: Research Method.
Cloning a Gene of Interest in a Plasmid Cloning Vector
Fig. 18-4a, p. 375

11. Introduce recombinant molecules into bacterial cells; each bacterium
Inserted genomic
DNA fragment
Recombinant
DNA molecules
Introduce recombinant molecules
into bacterial cells; each bacterium
receives a different plasmid. As the
bacteria grow and divide, the
recombinant plasmids replicate,
thereby amplifying the piece of DNA
inserted into the plasmid. 4
Bacterium
Bacterial
chromosome
Progeny
bacteria
Figure 18.2
Overview of cloning DNA fragments in a bacterial plasmid.
Identify the bacterium containing
the plasmid with the gene of interest
inserted into it. Grow that bacterium
in culture to produce large amounts
of the plasmid for experiments with
the gene of interest. 5
Fig. 18-2b, p. 373

12. Recombinant DNA
Break cells and use restriction enzyme to isolate DNA of interest (prokaryotic or eukaryotic)
Insert into plasmid (recombination)
Transform into bacteria (replication)
Not very efficient, so for the third step (isolation)- you need to have engineered a way to find the bacteria of interest

13. Four possibilities
1. Desired outcome: plasmid, lac+ broken, gene of interest inserted
2. Bacteria transformed with plasmid, but wrong gene inserted
3. Bacteria transformed with plasmid only- no gene at all inserted
4. Bacteria not transformed

14. Inserted DNA fragments with gene of interest
Inserted DNA fragment without gene of interest
Resealed plasmid cloning vector with no inserted DNA fragment
Recombinant plasmids
Nonrecombinant plasmid
Figure 18.4: Research Method.
Cloning a Gene of Interest in a Plasmid Cloning Vector
Fig. 18-4b, p. 375

15. Recombinant DNA
Insert into special screening plasmid- which contains the same restriction enzyme site used above, located in a lacZ gene.
For recombination screening the lacZ gene is broken successfully, it will be white. If not, it will be blue.
The plasmid also contain ampicillin resistance
If transformation worked, the bacteria will grow on plates containing ampicillin. Those who were not transformed will not grow.

16. Untransformed bacterium containing ampicillin.
Bacteria not transformed
with a plasmid
Bacteria transformed with plasmids
Selection:
Transformed bacteria grow on medium containing ampicillin because of ampR gene on plasmid.
Untransformed bacterium
cannot grow on medium
containing ampicillin.
Screening:
Blue colony contains bacteria with a non-recombinant plasmid; that is, the lacZ+
gene is intact.
Plate containing
ampicillin and X-gal
Figure 18.4: Research Method.
Cloning a Gene of Interest in a Plasmid Cloning Vector
White colony contains bacteria with a recombinant plasmid; that is, the vector with an inserted DNA fragment. Once the white colony with the gene
of interest is identified, it can be grown in culture to produce large quantities of the plasmid.
Fig. 18-4c, p. 375

17. DNA Hybridization
Uses nucleic acid probe to identify gene of interest in set of clones
Probe has tag for detection
Identified colony produces large quantities of cloned gene

18. containing ampicillin Bacterial colony
Culture medium
containing ampicillin
Bacterial
colony
Filter paper
Figure 18.5: Research Method.
DNA Hybridization to Identify a DNA Sequence of Interest
Replica of bacterial
colonies
Filter paper
Fig. 18-5a, p. 377

19. Labeled probe (single stranded) Plasmid DNA (single stranded) Bag
Labeled single-stranded DNA
probe for the
gene of interest
Bag
Filter
Hybridization has occurred between the labeled probe and the plasmids released from the bacteria in this colony. The hybridization is detected in subsequent steps.
Figure 18.5: Research Method.
DNA Hybridization to Identify a DNA Sequence of Interest
Fig. 18-5b, p. 377

20. Corresponds to Developed one colony on photographic master plate film
Figure 18.5: Research Method.
DNA Hybridization to Identify a DNA Sequence of Interest
Original master plate
Fig. 18-5c, p. 377

21. 4 Possibilities Outcome AMP LAC PROBE Right Gene yes No Yes Wrong Gene
Plasmid only
n/a
No Plasmid

22. Restriction Endonuclease?
How else do we use
Restriction Endonuclease?

23. RFLPs Restriction fragment length polymorphisms
DNA sequence length changes due to varying restriction sites from same region of genome
Sickle cell anemia has RFLPs
Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs
Alternative is PCR and electrophoresis

24. β-Globin gene 175 bp 201 bp Normal allele MstII MstII MstII 376 bp
Sickle-cell
mutant allele
Figure 18.8: Restriction site differences between the normal and sickle-cell mutant alleles of the β-globin gene.
The figure shows a DNA segment that can be used as a probe to identify these alleles in subsequent analysis (see Figure 18.9).
MstII
MstII
Region of probe
used to screen for
sickle-cell mutation
Fig. 18-8, p. 381

25. DNA Fingerprinting Distinguishes between individuals
Uses PCR at multiple loci within genome
Each locus heterozygous or homzygous for short tandem repeats (STR)
PCR amplifies DNA from STR
Number of gel electrophoresis bands shows amplified STR alleles
13 loci commonly used in human DNA fingerprinting

26. Forensics and Ancestry
Forensics compares DNA fingerprint from sample to suspect or victim
Usually reported as probability DNA came from random individual
Common alleles between children and parents used in paternity tests
Same principle used to determine evolutionary relationships between species

27. a. Alleles at an STR locus
Left PCR primer
DNA
9 repeats
Right PCR primer
3 different
alleles
11 repeats
Figure 18.10: Using PCR to obtain a DNA fingerprint for an STR locus.
(a) Three alleles of the STR locus with 9, 11, and 15 copies of the tandemly repeated sequence. The arrows indicate where left and right PCR primers can bind to amplify the STR locus.
15 repeats
Fig a, p. 383

28. b. DNA fingerprint analysis of the STR locus by PCR
Cells of
three
individuals
Extract genomic DNA
and use specific
primers to amplify
the STR locus using
the PCR.
Anyalyze PCR product by gel electrophoresis A B C
Positions corresponding to alleles of STR locus
Figure 18.10: Using PCR to obtain a DNA fingerprint for an STR locus.
(b) DNA fingerprint analysis of the STR locus by PCR. 15 11 9
11,11
15,9
11,9
Fig b, p. 383

29. Genetic Engineering Transgenic organisms
Modified to contain genes from external source
Expression vector has promoter in plasmid for production of transgenic proteins in E. coli
Example: Insulin
Protocols to reduce risk of escape

30. Animal Genetic Engineering
Transgenic animals used in research, correcting genetic disorders, and protein production
Germ-line cell transgenes can be passed to offspring (somatic can not)
Embryonic germ-line cells cultured in quantity, made into sperm or eggs
Stem cells

31. Germ-line cells derived from mouse embryo
Transgene
Cell with
transgene
Figure 18.11: Research Method.
Introduction of Genes into Mouse Embryos Using Embryonic Germ-Line Cells
Pure population of
transgenic cells
Fig a, p. 385

32. Mice have transgenic cells in body regions including germ line
Figure 18.11: Research Method.
Introduction of Genes into Mouse Embryos Using Embryonic Germ-Line Cells
Mice have transgenic cells in
body regions including germ line
Genetically engineered
offspring—all cells transgenic
Fig b, p. 385

33. Gene Therapy Attempts to correct genetic disorders
Germ-line gene therapy can’t be used on humans
Somatic gene therapy used in humans
Mixed results in humans
Successes for adenosine deaminase deficiency (bubble kid) and sickle-cell
Deaths from immune response and leukemia-like conditions

34. Animal Genetic Engineering
“Pharm” animals produce proteins for humans
Usually produced in milk for harmless extraction
Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell
Low cloning success rate
Increased health defects in clones
Gene expression regulation abnormal

35. Learning Objectives Define Clone and DNA Cloning
List the three steps of production of recombinant DNA
Describe the characteristics and uses of a restriction endonuclease
Diagram the process of identifying a transformed bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

36. Learning Objectives Explain the uses of RFLPs
Describe the process of producing a transgenic organism, and explain its usefulness