CHAPTER 9 DNA Technologies

– DNA cloning techniques – Expression of recombinant proteins – DNA analysis methods – DNA microarray technology

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Recombinant DNA

• Artificially created DNA that combines sequences that do not occur together in the nature

• Basis of much of the modern molecular biology – Molecular cloning of genes – Over-expression of proteins – Transgenic food, animals …

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DNA Cloning • Organism cloning

– Creation of identical copies of an organism

• DNA cloning – Creation of identical copies of a piece of DNA (gene) – Isolate a specific gene from the source organism and

amplify it in the target organism

• Basic steps – Cut the source DNA at the boundaries of the gene – Select a suitable carrier DNA (vector) – Insert the gene into the vector – Insert the recombinant vector into host cell – Let the host produce multiple copies of recombinant DNA

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DNA Cloning

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Restriction Endonucleases

• Cleave DNA phosphodiester bonds at specific sequences • Common in bacteria: Eliminates infectious viral DNA • Some make staggered cuts: Sticky ends • Some make straight cuts: Blunt ends • Large number are known: Commercially available

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Cloning Vectors • Plasmids

– Circular DNA molecules that are separate from the bacterial genomic DNA

– Can replicate autonomously • Origins of replication for use in bacteria and/or yeast

– Carry antibiotic resistance genes – Allows cloning of DNA up to 15,000 bp

• To clone whole chromosomes (up to 300,000 bp)

– Bacterial Artificial Chromosome (BAC) • For use in bacteria

– Yeast Artificial Chromosome (YAC) • For use in yeast

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• Enzyme that covalently joins two DNA fragments – Normally function in DNA repair – Human DNA ligase uses ATP – Bacterial DNA ligase uses NAD

DNA Ligase

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Antibiotic Selection

• Antibiotics, such as penicillin and ampicillin, kill bacteria

• Plasmids can carry genes that give host bacterium a resistance against antibiotics

• Allows growth (selection) of bacteria that have taken up the plasmid

NH

S

N

N

OO

H

OOH

HH

Ampicillin 13

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Identification of Empty Plasmids

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Separation of DNA by Electrophoresis

• Negatively charged DNA migrates to the anode in the presence of an electric field

• Agarose gel hinders the mobility of DNA molecules • Mobility depends on the size and the shape

– Small molecules faster – Compact molecules faster

• Practical use – DNA analysis – DNA purification – DNA-protein interaction studies

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Expression of Cloned Genes

• We want to study the protein product of the gene • Special plasmids, called expression vectors, contain

sequences that allow transcription of the inserted gene • Expression vectors differ from cloning vectors by having:

– Promoter sequences – Operator sequences – Code for ribosome binding site – Transcription termination sequences

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Site-Directed Mutagenesis

• Understanding the function of proteins often requires that a specific amino acid residue be mutated

• To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene

• Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA

• Mutated plasmids are always sequenced to confirm the desired (and only the desired) mutation is present

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Purification of Recombinant Genes • Purification of natural proteins is difficult • Recombinant proteins can be tagged for purification • The tag binds to the affinity resin, binding the

protein of interest to a purification column

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Polymerase Chain Reaction (PCR) • Used to amplify DNA in the test tube

– Can amplify regions of interest (genes) within linear DNA – Can amplify complete circular plasmids

• Mix together

– Target DNA, Primers, Nucleotides, Thermostable DNA polymerase

• Place the mixture into thermocycler – Melt DNA at about 95°C – Cool separated strands to about 50–60°C – Primers anneal to the target – Polymerase extends primers in 5’→3’ direction – After a round of elongation is done, repeat steps

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Repeat steps 1–3 many times: After 25 cycles DNA has been amplified about 106 fold

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DNA Fingerprinting • Humans have short sequences that repeat next to each other

– Short tandem repeats (STR)

• Differences in the number of repeats cause variations in the length of fragments that form when sample subjected to PCR using a primer specific for that region

• Fragment sizes can be determined by using a capillary gel • Multiple STR locations exist in the human genome • Allows matching “suspect” samples to known individuals • 13 well-studied locations are used in identifications

– Based on number of alleles seen at each location misidentification is less than 1 in 1018 (when good data is obtained)

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Adaptations to PCR

• Reverse Transcriptase PCR (RT-PCR) – Used to amplify RNA sequences – First step uses reverse transcriptase to convert RNA to DNA

• Quantitative PCR (Q-PCR) – Used to show quantitative differences in gene levels

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