1. Screening a Library for Clones Carrying a Gene of Interest
A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene
This process is called nucleic acid hybridization

2. For example, if the desired gene is
A probe can be synthesized that is complementary to the gene of interest
For example, if the desired gene is
– Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5

3. The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest
Once identified, the clone carrying the gene of interest can be cultured

4. Radioactively labeled probe molecules
Fig. 20-7
TECHNIQUE
Radioactively labeled probe molecules
Probe DNA
Gene of interest
Multiwell plates holding library
clones
Single-stranded DNA from cell
Film •
Figure 20.7 Detecting a specific DNA sequence by hybridizing with a nucleic acid probe
Nylon membrane
Nylon membrane
Location of DNA with the complementary sequence

5. Expressing Cloned Eukaryotic Genes
After a gene has been cloned, its protein product can be produced in larger amounts for research
Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

6. Bacterial Expression Systems
Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

7. Eukaryotic Cloning and Expression Systems
The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems
YACs behave normally in mitosis and can carry more DNA than a plasmid
Eukaryotic hosts can provide the post-translational modifications that many proteins require

8. One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes
Alternatively, scientists can inject DNA into cells using microscopically thin needles
Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

9. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

10. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8
TECHNIQUE 5 3
Target sequence
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Figure 20.8 The polymerase chain reaction (PCR)
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

11. TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a
Figure 20.8 The polymerase chain reaction (PCR)

12. Cycle 1 yields 2 molecules
Fig. 20-8b 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
Figure 20.8 The polymerase chain reaction (PCR)
New nucleo- tides

13. Cycle 2 yields 4 molecules
Fig. 20-8c
Cycle 2 yields 4
molecules
Figure 20.8 The polymerase chain reaction (PCR)

14. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8d
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence
Figure 20.8 The polymerase chain reaction (PCR)

15. DNA cloning allows researchers to
Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene
DNA cloning allows researchers to
Compare genes and alleles between individuals
Locate gene expression in a body
Determine the role of a gene in an organism
Several techniques are used to analyze the DNA of genes

16. Gel Electrophoresis and Southern Blotting
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size
A current is applied that causes charged molecules to move through the gel
Molecules are sorted into “bands” by their size
Video: Biotechnology Lab

17. Figure 20.9 Gel electrophoresis
TECHNIQUE
Mixture of DNA mol- ecules of different sizes
Power source –
Cathode
Anode +
Gel 1
Power source – +
Longer molecules 2
Shorter molecules
RESULTS
Figure 20.9 Gel electrophoresis

18. Mixture of DNA mol- ecules of different sizes
Fig. 20-9a
TECHNIQUE
Power source
Mixture of DNA mol- ecules of different sizes –
Cathode
Anode +
Gel 1
Power source
Figure 20.9 Gel electrophoresis – +
Longer molecules 2
Shorter molecules

19. Fig. 20-9b
RESULTS
Figure 20.9 Gel electrophoresis

20. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis
Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene
The procedure is also used to prepare pure samples of individual fragments

21. Fig
Normal -globin allele
Normal allele
Sickle-cell allele
175 bp
201 bp
Large fragment
DdeI
DdeI
DdeI
DdeI
Large fragment
Sickle-cell mutant -globin allele
376 bp
201 bp 175 bp
376 bp
Large fragment
DdeI
Figure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

22. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization
Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

23. Fig
TECHNIQUE
Heavy weight
Restriction fragments
DNA + restriction enzyme
I II III
Nitrocellulose membrane (blot)
Gel
Sponge
I Normal -globin allele
II Sickle-cell allele
III Heterozygote
Paper towels
Alkaline solution 1
Preparation of restriction fragments 2
Gel electrophoresis 3
DNA transfer (blotting)
Radioactively labeled probe for -globin gene
Figure Southern blotting of DNA fragments
Probe base-pairs with fragments
I II III
I II III
Fragment from sickle-cell -globin allele
Film over blot
Fragment from normal -globin allele
Nitrocellulose blot 4
Hybridization with radioactive probe 5
Probe detection

24. Restriction fragments DNA + restriction enzyme I II III
Fig a
TECHNIQUE
Heavy weight
Restriction fragments
DNA + restriction enzyme
I II III
Nitrocellulose membrane (blot)
Gel
Sponge
I Normal -globin allele
II Sickle-cell allele
III Heterozygote
Paper towels
Alkaline solution
Figure Southern blotting of DNA fragments 1
Preparation of restriction fragments 2
Gel electrophoresis 3
DNA transfer (blotting)

25. Radioactively labeled probe for -globin gene
Fig b
Radioactively labeled probe for -globin gene
Probe base-pairs with fragments
I II III
I II III
Fragment from sickle-cell -globin allele
Film over blot
Figure Southern blotting of DNA fragments
Fragment from normal -globin allele
Nitrocellulose blot 4
Hybridization with radioactive probe 5
Probe detection

26. DNA Sequencing
Relatively short DNA fragments can be sequenced by the dideoxy chain termination method
Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths
Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment
The DNA sequence can be read from the resulting spectrogram

27. Figure 20.12 Dideoxy chain termination method for sequencing DNA
TECHNIQUE
DNA (template strand)
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
DNA polymerase
dTTP
ddTTP
dGTP
ddGTP
DNA (template strand)
Labeled strands
Shortest
Longest
Direction of movement of strands
Longest labeled strand
Figure Dideoxy chain termination method for sequencing DNA
Detector
Laser
Shortest labeled strand
RESULTS
Last base of longest labeled strand
Last base of shortest labeled strand

28. Fig a
TECHNIQUE
DNA (template strand)
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
DNA polymerase
ddTTP
dGTP
ddGTP
Figure Dideoxy chain termination method for sequencing DNA

29. Direction of movement of strands Longest labeled strand
Fig b
TECHNIQUE
DNA (template strand)
Labeled strands
Shortest
Longest
Direction of movement of strands
Longest labeled strand
Detector
Figure Dideoxy chain termination method for sequencing DNA
Laser
Shortest labeled strand
RESULTS
Last base of longest labeled strand
Last base of shortest labeled strand

30. Analyzing Gene Expression
Nucleic acid probes can hybridize with mRNAs transcribed from a gene
Probes can be used to identify where or when a gene is transcribed in an organism