BIOL 3451: CHAPTER 20- RECOMBINANT DNA
67 Cards in this Set
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2 important tools in recombinant DNA technology
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restriction enzymes and DNA cloning vectors
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What are DNA libraries?
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Collections of cloned sequences
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What is a powerful technique for copying DNA?
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Polymerase chain reaction
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T or F: Molecular Techniques are used to analyze DNA
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TRUE
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What is the ultimate way to characterize DNA at the molecular level?
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DNA Sequencing
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What does Recombinant DNA mean?
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Different DNA molecules from different biological sources are combined.
- Sources are usually not found in nature
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What is the basic procedure for making Recombinant DNA?
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1. using Restriction enzymes--> to make specific DNA fragments
2. DNA fragments are then joined with a vector
3. Recombinant DNA is transferred to HOST cell to make multiple copies. (copies can be recovered from the host cell.
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CLONES
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are the recovered copies of a Recombinant DNA molecule.
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What can clones be used for?
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to study the structure and orientation of DNA
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What is Recombinant DNA technology used for?
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1. Isolate
2. Replicate "copy"
3. Analyze genes
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How does a restriction enzyme work to make restriction fragments?
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it binds to DNA's specific recognition sequence "restriction site" and cleaves off the DNA.
(look at Fig 20.1)
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What do most recognition sequences display?
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PALINDROME- a form of symmetry-nucleotide seq. reads the same on both strands when read in 5'-->3' direction.
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What does each restriction enzyme do?
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1.recognizes restriction sequence.
2.cuts DNA in cleavage pattern
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How long are most restriction sequences?
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4-6 nucleotides BUT some contain 8+
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What's the function of DNA ligase in Recombinant DNA?
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joins DNA fragments covalently to produce complete DNA
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VECTORS
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- carry DNA molecules that can copy DNA fragments in a host cell
-MUST :
-be able to replicate independently
-contain restriction enzyme sites to allow DNA fragments to be inserted.
-carry selectable gene marker- which …
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PLASMID
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-extrachromosomal (DNA is found out of nucleus)
-Double Stranded DNA molecule
-replicates independently from the chromosomes within bacterial cells.
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Name the 2 things plasmids used for DNA cloning usually contain
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a number of convenient restriction enzymes
a marker gene to select for its presence in the host cell
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What process introduces plasmids into bacteria?
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Transformation
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What are 2 techniques used in bacterial transformation (plasmid into bacteria)?
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1. cells are treated with calcium ions; DNA is heat shocked to be inserted into cells.
2.Electroporation-->high intensity pulse of electricity to move DNA into bacterial cells.
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What does cloning with a plasmid vector involve?
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1. both plasmid and DNA to be cloned are cut with same restriction enzyme.
2. DNA restriction fragments from the DNA to be cloned are added to the linearized vector when DNA ligase is present.
3. Recombinant DNA is made- then introduced to bacterial host cell by transformation.
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Selectable marker genes
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can easily identify Recombinant DNA
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What are some VERY EFFECTIVE selectable markers?
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Genes that provide resistance(fight/oppose) to antibiotics,like: ampicillin and lacZ genes.
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blue-white selection
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used to tell if a plasmid is inserted
-identifies cells that contain recombinant (bacterial cells=white)/non recombinant (cells= blue) DNA.
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Phage vectors- Lambda vector
(other cloning type of vector)
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-foreign DNA can replace the central 3rd of lambda phage with out affecting the ability to infect cells and replicate
-can carry up to 45kb of cloned DNA
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What are 2 other examples of vectors that can be used to clone large fragments of DNA?
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1. Bacterial artificial chromosomes (BAC's)
2. Yeast artificial chromosomes (YAC's)
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Bacterial Artificial Chromosomes
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- usually very large BUT copy low numbers (1-2 copies of bacterial cell) plasmids.
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Expression Vectors
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made to produce a gene of interest that will make large amounts of encoded protein in host cell.
-available for both Prokaryotic & Eukaryotic host cells
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T or F: Besides bacteria and yeast being able to serve as hosts for recombinant DNA, plant and animals can't?
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FALSE: Plant and animal cells can serve as host cells for recombinant DNA too.
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rhizobium radiobacter (formerly known as Agrobacterium tumefaciens)
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-soil bacterium which infects cells and produces tumors
-can be used to transform plant cells
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Rhizobium Radiobacter
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Contains:
1. Ti plasmid (tumor inducing)
-tumor inducing gene is removed so rec. vector doesn't produce tumors
2. T-DNA: segment in Ti-plasmid carrying bacteria to infect plant cells- is transferred into genome of host cell
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Recombinant vector of Rhizobium radiobacter
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vector +mixed plant cells enters inside the cell & foreign DNA is inserted into Genome
-Plant cells can be grown in tissue culture and regenerate a mature plant carrying foreign gene.
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Why is Yeast used as a host for DNA cloning in Eukaryotic genes?
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- grown/manipulated easily
-intense study of genetics
-entire genome has been sequenced
-able to posttranslationally modify eukaryotic proteins
-its safe
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DNA libraries
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Collections of:
cloned sequences- usually come from single source (tissue type, cell type, or single individual.
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Genomic Libraries
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contains at least 1 copy of all the sequences in the genome of interest
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How are genomic libraries constructed?
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They are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors
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In genomic libraries
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- YAC's were used to accomodate large sizes of DNA needed to span 3 billion ppb of DNA
-whole-genome shotgun cloning approaches and next-generation sequencing are replacing traditional sequencing.
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complementary DNA libraries (cDNA)
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contain complementary DNA copies made from mRNAs present in a cell population
-represents genes that are transcriptionally active at the same time cells were collected from mRNA isolations.
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How is a cDNA made?
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1.mRNA from cells are isolated
2.reverse transcriptase is used to make complementary DNA
3.cDNA molecules are cloned into vector
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PCR (RT-PCR) in reverse transcriptase
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-can make cDNA from mRNA by:
1. making a ssDNA cDNA copy of the mRNAs using reverse transcriptase.
2. using PCR to copy SS-DNA into DS-DNA
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cDNA libraries
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provide:
-instant catalog of all genes active in a cell at a specific time
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Library screening
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used to sort through a library and isolate specific genes of interest.
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Probes
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used to screen a library to recover clones of a specific gene.
-it is any DNA or RNA sequence that is complementary to target gene of sequence to be identified.
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To screen a plasmid library
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-clones from the library are grown on agar plates to make colonies
-colonies are screened by:
-transferring bacterial colonies from the plate to a filter
-filter is hybridized with nucleic acid probe to the DNA sequence of interest.
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Results of plasmid screening
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- colony corresponds to the probe identified on the filter and it is recovered
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Phage library
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screened by plaque hybridization
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Polymerase chain reaction (PAR)
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copies specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small amounts.
-its rapid DNA cloning method
-doesn't require host cell
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Process of cloning DS-DNA
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DS-DNA is put in a tube with DNA polymerase, Mg^2+ & 4 deoxyribonucleoside triphosphates
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What does PCR require?
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2 oligonucleotide primers( short single stranded sequences)
-one complement to 5' end of one strand of the target DNA to be amp'd
-another complementary to 3' of other strand.
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How are the complementary strands made?
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primers harden to denatured DNA complementary strands are produced by heat-stable DNA polymerase
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3 steps for PCR
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1.denaturation
2.primer annealing
3.extension
-uses thermocycler to amplify the DNA
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What is a limitation of PCR?
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info about nucleotide sequence must be known in order to produce primer
-contamination from other sources can cause a problem
-can't amplify long segments of DNA
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PCR
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-very useful in identifying mutations in genetic disorders.
-techniques are advantageous when studying samples from single cells, fossils, or crime scene evidence- hair strand, saliva are collected.
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Reverse Transcription PCR (RT-PCR)
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used to study gene expression by studying mRNA replication by cells or tissues
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Quantitative real-time PCR (q-PCR)
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allows researchers to quantify amplification reactions as they occur in real time.
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what does q-PCR use?
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-procedure uses SYBR green dye and TaqMan probes, which contain 2 dyes.
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What are some molecular techniques for analyzing DNA?
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1.Restriction mapping
2.Southern blot
3.Northern blot
4. FISH
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Restriction Map
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determines restriction sites number, order, and distance between between them on cloned DNA segments.
-provides length of clone inserted and location of restriction sites
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How where restriction maps created?
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cutting DNA with different restriction enzymes and separating DNA fragments by gel electrophoresis.
-small fragments move farthest in gel
-can be seen when stained with ethidium bromide and illuminated by UV light
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Southern Blot
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-identifies clones in a library that contain a given DNA sequence
-characterizes size of fragments from restriction digest.
-used to determine whether a clone contains all or part of a gene
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What are the 2 components of southern blots?
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gel- separates DNA fragments
labeled probes used for electrophoresis and hybridization.
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Northern Blot analysis
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-determines if a gene is actively being expressed in cell or tissue
-used to study patterns of gene expression in: embryonic tissue, cancer, and genetic disorders.
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FISH- Fluorescent in situ hybridization
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-probe is hybridized directly to chromosome OR RNA without blotting.
-can be carried out on slide or in situ in tissue sections or entire organisms.
-helpful when studying embryos.
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DNA sequencing ultimate way to characterize DNA structure at molecular level
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1. Sanger sequencing - dideoxynucleotide chain termination sequence= most common
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1. Sanger sequencing - dideoxynucleotide chain termination sequence= most common
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developed by SANGER
-involves- addition of small amount of 1 dideoxynucleotide- causes DNA synthesis to end.
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Computer- automated high-throughput DNA sequencing
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-Generates large amounts of DNA
-enabled rapid progress in human genome
-uses fluorescent dye-labeled dideoxynucleotides.
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Next-Generation sequencing
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-outdates automated high throughput DNA sequencing.
-it's cheaper.
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BIOL 3451: CHAPTER 20- RECOMBINANT DNA