DNA Technology Flashcards
DNA cloning
. Cell-based procedure for copying DNA
. Studies to understand genetic involvement in disease processes or used for production of therapeutic proteins
. Done by cutting a DNA sequence out of genome via restriction endonucleases, inserting it into vector (bacterial plasmid) to create recombinant DNA in cell-based system
Plasmids
. Circular extrachromosomal DNA found in bacterial cells that replicate independently
Restriction endonucleases
. Hydrolyze human DNA at palindromic sequences which occur randomly throughout genome to generate multiple DNA fragments w/ no unpaired nucleotides on their ends (blunt ends) or short sequence of 2-4 unpaired nucleotides (single stranded DNA) on each end (sticky ends)
Cloning vectors
. Fragments made by restriction endonucleases are inserted into vector to permit transfer into cell for copying
. Weak (base pairing for DNA w/ sticky ends) binds to digested DNA to plasmid and covalent joining to form recombinant DNA molecule via DNA ligase
.
Important characteristic of plasmids used for cloning
. Presence of origin of replication site
. Single recognition sire for at least 1 restriction endonuclease (so plasmid can be opened to allow DNA insertion to be cloned, more than one site would cause plasmid being cut)
. At least 1 gene conferring a means to identify bacteria containing the vector
Potential cloning vectors
. Antibiotic resistance . Cosmids . Bacterial artificial chromosomes . Yeast articular chromosomes . Lambda phage
Genomic DNA libraries
. Contain entire genomic DNA including exons, introns, gene regulatory sequences , and non-coding DNA
. Plasmids prepared from a digest of genomic DNA isolated from an organism
Complementary DNA (cDNA) libraries
. Contain DNA only from exons of expressed genes
. Plasma prepared from digest of reverse-transcribed cellular messenger RNA
How to identify the bacterial clone containing the gene of interest in a DNA library
. Library is probed w/ labeled oligonucleotide complementary to the region of interest
. Hybridization (complementary base-pairing) of the labeled probe to the corresponding single stranded DNA sequence allows identification and isolation of cloned gene for further study
Production of therapeutic proteins
. Needs expression vector
. Only cDNA (not genomic) can be expressed in bacterial cells bc they can’t splice
. Coding sequences of cDNA is joined to strong bacterial promoter
. 5’-UTR of transcript contains bacterial ribosome-binding sequence
. Expression vector contains transcription/translation termination signals and protein processes (some have genes for protease inhibitors to enhance yield)
. Proteins needed posttranslational modifications are produced in eukaryotic cells
Shine-Dalgarno sequence
. Bacterial ribosome binding sequence in 5’-UTR transcript
Why can’t proteins needing posttranslational modifications be produced in prokaryotes?
. Unable to perform glycosylation or other modifications in cell
Therapeutic proteins produced via DNA technology and their functions
. Humulin: insulin . Lantus: long-acting insulin glargine . Recombivax HB: hep B vaccine . Recombinate: factor VIII (hemophilia A) . Epogen (EPO)
Advantages of therapeutic proteins produced via DNA technology
. Production of human form of therapeutic protein lessens antigenicity
. Less risk for contamination of therapeutic protein w/ infectious agents
. Relatively low cost for large-scale production
Taql
. Cleaves into 4 base cohesive ends
. Sticky ends
Haell
. Four base, blunt ends
Polymerase chain reaction
. Used for rapid amplification of DNA
. DNA heated to separate to single strands
. DNA primers complementary to nucleotide sequences flanking region of interest added
. DNA cools and anneals
. Heat stable DNA Pol and excess dNTPs added for new complementary DNA strand synthesis
. Cycle repeated 20-30 times
PCR applications
. Detection of infectious agents
. Prenatal genetic diagnoses (nested primers)
. Multiplex PCR to screen for disease caused by multiple mutations
. Diagnosis of point mutations
DNA gel electrophoresis
. DNA (net neg. charge) migrates towards anode
. Smaller DNA fragments migrate further
Prenatal genetic diagnoses using nest primers in PCR
. 2 rounds PCR
. 1st w/ outer primers
. 2nd amplifies product of 1st round through inner primers
. Ability to detect very small amounts of DNA
. Enhanced sensitivity to detect very small amounts of genetic material
Multiplex PCR for muscular dystrophy
. . Disease caused by numerous mutations in DMD gene
. Many mutations leading to disease cause complete loss of an exon
. Multiplex protocol uses simultaneous amplification w/ primers specific for 18 exons known to be assoc. w/ deletion hot spots
. Multiplex detects 90-98% of all disease-causing deletions
Nucleic acid hybridization
. Detection of specific DNA sequences relies upon ability of labeled fragments of ssDNA (termed probes) complementary to sequence of interest
. Sequence-specific probes assoc. w/ sequence of interest following its separation into ssDNA (by alkaline denaturation)
. Binding of probe to sequence of interest can then be visualized by analytical methods
In situ hybridization
. Labeled probe is added to chromosome preparation whose DNA has been denatured in place
. Hybridization (pairing probe w/ complementary DNA sequence) is allowed to occur
. Excess (non-hybridized) probe removed and physical site of hybridization of the labeled probe is detected
Fluorescence in situ hybridization
. Probes labeled w/ fluorescent molecules
. Versatile and diagnostic tool
. Used in cytogenetic
Southern blotting
. Detects dNA fragments containing specific sequences
. Extract DNA from cells
. Restriction enzyme digestion of DNA -> DNA fragments
. Separation of DNA fragments by size via electrophoresis
. Identification of specific DNA fragments by probe-hybridization
Northern blot
. Southern blot procedure but with RNA
Western blot
Southern blot procedure but w/ protein
. Used as diagnostic for HIV
Restriction fragment length polymorphism
. Genetic variants from mutations that cause either gain or loss (more common) of restriction site
. Result in change in DNA fragment size following restriction digestion that is detectable via southern blotting
. Analysis used diagnostically if RFLP is closely assoc. w/ disease-causing mutation or mutation that gives rise to disease is the same as that resulting in polymorphism
Polymorphism in repetitive DNA
. Present in micostellite sequences of genome (variable numbers of tandem repeats/VNTR)
. Detected via RFLP analysis
. VNTR unique to individual
VNTR uses
. Forensics to identify people in crimes
. Inherited as simple Mendelian traits
Allele-specific oligonucleotides (ASOS)
. Probes specific to unique DNA sequence
. Used in PCR analysis to determine genotype of individual by testing whether DNA sample from individual is amplifiable by wild type, mutant, or both
Dot blotting
. Rapid variant of ASO analysis
. DNA sample denatured and applied directly to nitrocellulose membrane
. Dots of denatured DNA is the probed (blotted) w/ radioactive labeled ASOS complementary to both wild type and mutant genes
. Simple, rapid, inexpensive
Tests used to analyze gene expression
. Northern and western blots
. Microarrays
. ELISA
. Proteomics
Gene therapy
. Potential to treat number of genetic diseases
