DNA Technology Flashcards
DNA technology permits
Manipulation and analysis of DNA, synthesis and mutation of genes, clone and express genes to produce proteins, locate gene mutations, detect carriers of genetic disease
Enzymes used in DNA technology
Restriction endonucleases, DNA ligase, DNA polymerase, Reverse transcriptase
Restriction endonucleases
Restriction enzymes, cut double-stranded DNA into smaller fragments, cut at specific sequence- restriction site, isolated from bacteria
Restriction site
Specific sequence of bases cleaved by a restriction enzyme, most commonly 4-6 base pairs long, many are palindromic (both strands have same sequence when read 5’ to 3’)
Sticky ends
Restriction enzyme cuts at palindromic restriction site, leaves staggered ends of the DNA
Blunt ends
Restriction enzymes cuts evenly through both strands of DNA
DNA ligases
Join together fragments of DNA, in vivo they repair single-stranded breaks, in vitro then join fragments of double-stranded DNA
Ligation
Joining together DNA molecules, more efficient between sticky ends, can add linkers to blunt ends to make them sticky
DNA polymerases
Makes copies of DNA, synthesize a single new strand complementary to an existing single-stranded DNA template, most require a double-stranded primer
Reverse transcriptase
Copies RNA onto DNA, synthesizes a DNA strand complementary to an RNA template, used in production of complementary DNA (cDNA), required for production of recombinant eukaryotic proteins in prokaryotes
Analyzing the result of a DNA reaction
Reaction products are separated by gel electrophoresis, DNA molecules move according to their size, smaller the molecule, the faster it moves, bands are visualized
Separation of products by gel electrophoresis
DNA loaded into a well cut out of the gel, DNA separates into bands of different-sized fragments
Estimation of product size
Size markers- mix of DNA fragments of known, different lengths
Hybridization techniques
Techniques involving annealing of two single strands of complementary base sequence, involve use of probe to identify or select specific DNA fragments
Probe
Short single-stranded nucleotide of known sequence, usually labelled to permit identification
Southern blotting
Identification of DNA fragments through their ability to hybridize with a complementary probe
Northern blotting
Identification of RNA fragments
Western blotting
Identification of proteins through their ability to bind to specific antibodies
Applications of southern blotting
Identifying genes, diagnosis of genetic diseases, carrier detection, DNA fingerprinting
DNA profiling (fingerprinting)
Original method used probes that annealed to DNA regions comprising repeated blocks of bases, number of repeats varies from one individual to another
Minisatellite DNA
Comprises variable number tandem repeats (VNTRs)
Microsatellite DNA
Compromises short tandem repeat polymorphism (STRPs)
DNA sequencing
Determination of the sequence of bases in a DNA molecule, Maxam-Gilbert chemical method, chain termination or dideoxy method, automated sequencing using fluorescence
DNA sequencing protocol
4 sequencing reactions are set up, one for each base, each reaction is set up to synthesize a nucleotide chain complementary to the chain under analysis, all reaction components are added to each reaction plus a small amount of ddNTP
Automated DNA sequencing
Based on Sanger method, employs fluorescent labeling, different fluorolabel attached to each ddNTP, all 4 reactions performed in one tube and electrophoresis performed in one lane of the gel
Polymerase chain reaction (PCR)
In vitro method for producing large amounts of DNA from a target sequence, requires only minute amount of sample to be amplified, can produce a million copies in 2-3 hours
PCR reaction components
Target DNA template, deoxynucleotide mixture (dATP, dTTP, dCTP, dTTP), heat stable DNA polymerase, oligonucleotide primers complementary to ends of region to be amplified
Steps of PCR
Heat separates strands, add synthetic oligonucleotide primers, cool, add thermostable DNA polymerase to catalyze DNA synthesis, repeat, DNA synthesis catalyzed by thermostable DNA polymerase, target sequence amplified 10^6 fold after 25 cycles
Advantages of PCR
Size and condition of sample, speed
Disadvantages of PCR
Some sequence knowledge required, size limit for target DNA, contamination
Uses of PCR
Accelerating and facilitating genetic engineering, diagnosing genetic diseases, identifying carriers of genetic diseases, detecting presence of infectious organisms, DNA profiling
Vectors
Vehicles that carry DNA fragments into cells- plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), vectors for gene therapy (viruses)
Transduction
Phage mediated gene transfer into a prokaryotic cell
Transformation
Non-phage mediated gene transfer into prokaryotic cell
Transfection
Gene transfer into a eukaryotic cell
Formation of recombinant DNA molecule
Add gene to vector with DNA ligase, forming a recombinant DNA molecule
Clone
Group of organisms, cells, or molecules all originating from a single individual
Clone a gene
Isolate the DNA that encodes the gene in a pure, readily reproducible form, can be used for amplifying DNA or production of the encoded protein from cloned DNA
DNA libraries
Large collection of recombinant DNA clones, in which the DNA has been inserted into a vector
Genomic library
A collection of DNA fragments from an organism’s entire genome cloned into bacterial colonies, contains all genes for an organism
cDNA library
Collection of cDNA fragments produced from all the mRNA present in a particular tissue type, contains only cDNAs corresponding to proteins expressed in the cell type from which the library was made
Screening a genomic library
Selecting a colony that contains a specific individual gene, hybridization with an oligonucleotide probe
Screening a cDNA library
Polymerase chain reaction, hybridization with an oligonucleotide probe, immunological screening
Medical applications of DNA technology
Diagnosis of genetic diseases, carrier detection, detection of infectious organisms, production of therapeutic proteins, production of DNA vaccines, gene therapy
DNA techniques in diagnosis
Most involve preliminary amplification by PCR- restriction fragment length polymorphism (RFLP) analysis, direct PCR analysis, allele-specific oligonucleotide (ASO) hybridization, hybridization to microchip array
Detection of mutation by direct PCR analysis
PCR amplification of mutated region
Allele-specific PCR amplification (ARMS test)
PCR equivalent of ASO hybridization, achieves the same purpose in one PCR step, 2 sets of primers: 1st primer the same for both reactions, 2nd primer in 2 forms, normal and mutant, only exact complementary target sequence is amplified
PCR target size limitations
Routine use, not >4000 bases, disorders due to triplet nucleotide expansions, full mutation too large to amplify routinely
DNA microarrays
Used to detect mutations and to examine gene expression, normal and mutated ASOs robotically placed on glass slide, hybridized with fluorescently labeled DNA from patient, pattern analyzed by computer
Therapeutic proteins produced by recombinant DNA technology
Insulin, factor VIII, factor IX, human growth hormone, tissue plasminogen activator, interferon, erythropoietin, adenosine deaminase
Production of vaccines by recombinant DNA technology
Protein antigens can be produced completely free of the infectious agent, hepatitis B vaccine was first produced recombinant DNA vaccine
Gene therapy
To cure or prevent a disease by modifying an individual’s genes or their expression, somatic cell therapy or germline therapy
Somatic cell therapy- gene replacement therapy
Replacing a mutated gene that causes disease with a healthy gene
Somatic cell therapy- gene therapy for non-inherited diseases
Introducing a new gene to help fight a disease
Somatic cell therapy- gene-blocking therapy
Inactivating a mutated gene that is functioning improperly
Retrovirus vectors
Advantages- enters cells efficiently, integrates stably into host genome, targets only dividing cells
Disadvantages- limited insert size, integrates into host genome, targets only dividing cells
Adenovirus vectors
Advantages- enters cells efficiently, high expression of insert, does not integrate into host genome
Disadvantages- can elicit serious immune response, does not integrate into host genome
Adeno-associated virus vectors
Advantages- enters cells efficiently, elicits little or no immune response, integrates into host genome at specific site
Disadvantages- limited insert size, difficult to produce
Herpes simplex virus vectors
Advantages- can carry up to 20kb, prolonged activity, infects nerve cells efficiently
Disadvantages- can cause immune response
Liposome vectors
Advantages- can accept large inserts, no immune response
Disadvantages- inefficient cell entry, no integration into host genome, can be toxic
Naked DNA vectors
Advantages- can accept large inserts, no immune response
Disadvantages- very inefficient cell entry, no integration into host genome
Viral vs non-viral vectors
Non-viral vectors are better suited for ex vivo gene therapy than in vivo gene therapy
Ex vivo gene therapy
Patient’s cells are extracted, manipulated outside the body, and re-introduced, used for SCID, familial hypercholesterolemia, Gaucher disease, malignant melanoma, leukemia
In vivo gene therapy
Used for cystic fibrosis, various cancers (brain tumors, ovarian cancer, neck cancer), hemophilia B, Duchenne muscular dystrophy
Success with human gene therapy
SCID, malignant melanoma, Leber’s congenital amaurosis, hemophilia B, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Parkinson disease, cystic fibrosis
Gene therapy for lipoprotein lipase deficiency
Glybera approved as treatment, delivers lipoprotein lipase in an adeno-associated virus (AAV) vector, one time intramuscular injection
Transgenic animals and plants
Organisms with foreign DNA introduced in the genome, can increase growth rate, disease resistance, produce nutritional supplements
Compounds produced in pharm animals
Human hemoglobin, human tissue plasminogen activator (TPA)
