Chapter 17 Flashcards
Recombinant DNA Technology
Recombinant DNA
Recombinant DNA
- Joining of DNA molecules
- Produced by artificially joining DNA from different biological sources not found together in nature
Clones
- recovered copies of recombinant DNA molecule
Recombinant DNA technology
- used to isolate, replicate, and analyze genes
Restriction Enzymes and Cloning Vectors
- Two important tools used to construct and amplify DNA molecules–DNA-cutting enzymes: restriction enzymes–DNA cloning vectors
Restriction Enzymes Cut DNA at Specific Sites
Restriction enzymes
- Produced by bacteria as defense mechanism against bacteriophage
- DNA-cutting enzymes
- Bind to DNA at specific recognition sequence (restriction site) and cleave DNA to produce restriction fragments
- Enzyme cleaves both strands of DNA (digestion)
Recognition Sequences
Palindrome
–Symmetry exhibited by recognition sequences (Nucleotide sequence reads same on both strands).
–Restriction enzymes cut DNA in characteristic cleavage pattern (Figure17-1).
–Sticky ends (cohesive ends): fragments produced with overhangs
–Blunt ends: fragments produced with double- stranded ends
DNA Ligase
DNA ligase
–DNA fragments will seal phosphodiester backbone.
–Joins restriction fragments covalently to produce intact DNA molecules
DNA Vectors Accept and Replicate DNA Molecules to Be Cloned
Vectors: carry DNA molecules
- can replicate cloned DNA fragments in hosts cells
- must be able to replicate independently
- have several restriction enzymes sites to allow insertion of DNA fragment
- carry selectable gene marker to distinguish host cells that have taken them up from those that have not
Plasmids
Plasmids used in DNA cloning
- genetically modified bacterial plasmids - first vectors developed
- genetically engineered to contain: number of convenient restriction sites and a marker gene to select for presence in host cell
Transformation
Plasmids are introduced into bacteria via transformation
- 2 main techniques
1. using ca ions and brief heat shock to pulse DNA into cells
2. electroporation: a brief but high intensity pulse of electricity to move DNA into bacterial cells
DNA cloning
- Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme
- DNA restriction fragments from DNA to be cloned are added to linearized vector in the presence of DNA ligase
- Recombinant DNA is produced and introduced into bacterial host cells by transformation
Selection markers
selectable marker genes
- genes provide resistance to antibiotics
Blue-white selection
- used to identify cells containing recombinant and non-recombinant DNA
- plasmid contains lacZ gene which encodes B-galactosidase
blue-white screening mechanism
Blue - bacterial cells with functional lacZ gene carrying a non recombinant plasmid
White - bacterial cells with recombinant plasmid
Other types of cloning vectors
- phage vector systems
- Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)
phage vector systems
- among earliest vector used in addition to plasmids
- central third of lambda phage vectors can be replaced with foreign DNA without affecting ability to infect cells and replicate
Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)
- Vectors used to clone large fragments of DNA
- BACs are generally very large and circular.
- YACs are linear and have telomeres at each end
DNA libraries
- represent a collection of cloned DNA
- Two main types: genomic and complementary (cDNA)
Genomic library
- contains at least 1 copy of of all sequences in genome of interest
- constructed by cutting genomic DNA with restriction enzyme and ligating fragments into vectors
cDNA library
- contain complimentary DNA copies made from mRNAs present in cell population
- represents genes active transcriptionally at the time cells were collected for mRNA isolation
Specific Genes can be recovered from a library by screening
Library screening
- used to sort through library and isolate specific gene of interest
Probes
- used to screen library and recover clones of specific gene
- a probe is any DNA or RNA sequence complementary to to target gene of sequence being identified
Screening a plasmid library
- clones from library are grown on agar to produce colonies
- colonies are screened by transferring bacterial colonies from plate to filter
- filter is hybridized with nucleic acid probe to DNA sequence of interest
- colony corresponding to probe identified on filter is recovered
The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA
(PCR)
–Rapid method of DNA cloning eliminates need to use host cells for cloning.
–Copies specific DNA sequence via in vitro reactions; can amplify target DNA sequences present in very small quantities
PCR process
PCR requires two primers
–Primers: short, single-stranded sequences, one complementary to 5’ end and another complementary to 3’ end
–DNA to be cloned is put in tube with DNA polymerase, Mg2+, and dNTPs.
–Primers anneal to denatured DNA.
–Complementary strands are synthesized by heat-stable DNA polymerase.
Steps of PCR
- Denaturation
- Primer annealing (hybridization/annealing)
- Extension
- Steps are repeated over and over using thermocycler to amplify DNA exponentially.
- DNA strand is doubled in each cycle.
- New strands along with old strand serve as templates in next cycle.
Limitation of PCR
- Some information about nucleotide sequence of target DNA is required to synthesize primer.
- Minor contamination from other sources can cause problems (e.g., skin cells from researcher).
–PCR cannot amplify long segments of DNA.
Applications of PCR
–Useful tool; most widely used technique in genetics and molecular biology
–Allows for screening of mutations involved in genetic disorders
–Location and nature of mutation can be determined quickly.
Varieties of PCR
Reverse transcription PCR (RT-PCR)
–Methodology for studying gene expression (mRNA production by cells or tissues)
–Reverse transcriptase is used to generate ds-cDNA.
Quantitative real-timePCR (qPCR)
–Real-time PCR allows researchers to quantify amplification reactions as they occur in real time.
Gene Targeting and Knockout Animal Models
Gene targeting
–Concept: to manipulate specific allele, locus, or base sequence and learn its function on gene of interest
–Manipulates specific gene in genome
Gene knockout
–Concept: to disrupt or eliminate specific gene/genes and see “what happens”
–Knockout mice have revolutionized research.
Creating knockout mice
–To make KO mice, construct a targeting vector; this creates segment of DNA for introduction into cell.
–Targeting vector then undergoes homologous recombination with gene of interest and renders it nonfunctional.
–Target vector has mutated a copy of gene of interest.
–Foreign DNA disrupts the reading frame and produces nonfunctional protein
Target Vectors Introduced into Cells
Embryonic (ES) stem cells
–Using ES cells, scientists introduce targeting vectors into cells via electroporation.
–ES cell takes in targeting vector, homologous recombination can occur.
–Recombinant ES cells are selected and injected into mouse embryo.
–Results: chimeras (Figure 17-14)
Transgenic animals: Knock-in animals
–Express or over express particular gene of interest (transgene)
–Vector with transgene can also be put into ES cells and injected into embryos.
–Allow for study of effects on appearance and function in mice
