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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

Fig. 20-9b RESULTS Figure 20.9 Gel electrophoresis

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

Fig. 20-10 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 20.10 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

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

Fig. 20-11 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 20.11 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

Restriction fragments DNA + restriction enzyme I II III Fig. 20-11a 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 20.11 Southern blotting of DNA fragments 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled probe for -globin gene Fig. 20-11b 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 20.11 Southern blotting of DNA fragments Fragment from normal -globin allele Nitrocellulose blot 4 Hybridization with radioactive probe 5 Probe detection

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

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 20.12 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

Fig. 20-12a TECHNIQUE DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP ddATP dCTP ddCTP dTTP DNA polymerase ddTTP dGTP ddGTP Figure 20.12 Dideoxy chain termination method for sequencing DNA

Direction of movement of strands Longest labeled strand Fig. 20-12b TECHNIQUE DNA (template strand) Labeled strands Shortest Longest Direction of movement of strands Longest labeled strand Detector Figure 20.12 Dideoxy chain termination method for sequencing DNA Laser Shortest labeled strand RESULTS Last base of longest labeled strand Last base of shortest labeled strand

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