CHAPTER 9 DNA Technologies
– DNA cloning techniques – Expression of recombinant proteins – DNA analysis methods – DNA microarray technology
1
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 …
2
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
3
DNA Cloning
4
5
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
6
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
7
8
9
10
• Enzyme that covalently joins two DNA fragments – Normally function in DNA repair – Human DNA ligase uses ATP – Bacterial DNA ligase uses NAD
DNA Ligase
11
12
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
14
Identification of Empty Plasmids
15
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
16
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
17
18
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
19
20
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
21
22
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
23
24
Repeat steps 1–3 many times: After 25 cycles DNA has been amplified about 106 fold
25
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)
26
27
28
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
29
30