Genetics: Chapter 17: Recombinant DNA Technology

Recombinant DNA

-DNA created by joining together pieces of DNA from different sources -techniques were developed to analyze it in the mid to late 1970s

Northern Blot

-RNA blotting -related to Western Blot

dNTP

-one less of oxygen

ddNTP

-two loss of oxygen

DNA Sequencing

-ultimate way to characterize DNA structure at the molecular level -most common method is dideoxynucleotide chain-termination sequencing or Sanger sequencing -since early 1990s,, largely been done through computer-automated Sanger reaction-based technology or computer-automated high-throughput DNA sequencing

Next-generation sequencing (NGS) technologies

-use state-of-the-art techniques to generate massive amounts of DNA sequence data at dramatically reduced costs per base

Molecular Techniques for Analyzing DNA

-used for analyzing genomic structure, expression, regulation, and function -one of first steps in characterizing a DNA clone is constructing a restriction map

Recombinant DNA Technology

-used to copy or clone DNA -allowed scientists to isolate and study specific DNA sequences -used to investigate the genomes of diverse species

Blue-White Selection

-used to identify cells containing recombinant or nonrecombinant DNA

Expression Vectors

-designed to ensure mRNA expression of a cloned gene with the purpose of producing large quantities of the encoded protein in a host cell -used to produce commercially valuable protein products from cloned genes

Dideocynucleotide Chain-Termination Sequencing or Sanger Sequencing

-developed by Sanger -double-stranded DNA to be sequenced is converted to single-strands to be used as templates for synthesizing a series of complementary strands -technique involves the addition of small amount of one modified deoxyribonucleotide called dideoxynucleotide, causing DNA synthesis to terminate -dramatic modifications have led to technologies that allow sequencing reactions to occur in a single tube in which each of the four ddNTPs is labeled with a different-color fluorescent dye -DNA fragments moving through the gel are scanned with a laser, stimulating the fluorescent dyes to emit different wavelengths of light, which a computer converts into DNA sequences -outdated when it comes to sequencing entire genomes

SOLiD (Supported Oligonucleotide Ligation and Detection)

-developed by company Applied Biosystems (ABI) -produces 6 gigabases of sequence data per run

Restriction Map

-establishes the number of, order of, and distances between restriction sites along a cloned segment of DNA -provides information about length of cloned insert and location of restriction sites within the clone when trying to reclone -created experimentally by cutting DNA with different restriction enzymes and separating DNA fragments by gel electrophoresis, which sepearates fragments by size -most are now created by using software to identify restriction enzyme cutting sites in sequenced DNA

Computer-automated high-throughput DNA Sequencing

-generates large amounts of sequence of DNA at greatly reduced costs -enabled rapid progress of human genome project

Flourescent in situ hybridization (FISH)

-involves hybridizing a probe directly to a chromosome or RNA without blotting -can be carried out with isolated chromosomes on a slide or in situ in tissue sections or entire organisms -especially helpful when embryos are used for various studies in developmental genetics -used to produce karyotypes to detect individual chromosomes or spectral karyotyping -detects deletions, translocations, duplications

DNA Vectors

-accept and replicate DNA molecules to be cloned -cloning vectors are carrier DNA molecules that accept DNA fragments, replicate inserted DNA fragments in a host cell such as bacteria, plant, or yeast -several key properties >>>contain several restriction sites that allow insertion of DNA fragments to be cloned >>>must be introduced into host cells to allow independent replication >>>should carry a selectable marker gene that distinguishes it from host cells. which cam be an antibiotic resistance gene or gene for an enzyme absent from host celll >>>many incorporate specific sequences that allow for sequencing inserted DNA

Quantitative Real-Time PCR (qPCR)

-allows researchers to quantify amplification reactions as they occur in real time without having to run a gel

Phage Vectors

-among earliest vectors used in addition to plasmids -include genetically modified strains of lambda phage vectors that can carry up to 45 kb of cloned DNA, which is more than twice in most plasmid vectors -from viruses that affect bacteria -"bacteria viruses" -carry larger fragments of DNA

Third Generation Sequencers

-available and will be widely used in next few years -provide direct sequencing of DNA in a single strand -use nanotechnology that cleaves off individual bases from single-stranded DNA fragment -does not require DNA amplification or flourescence tagging

Librariees enable scientists to...

-clone DNA and then identify individual genes in the library

DNA Libraries

-collections of cloned sequences -represent a collection of cloned DNA samples that contain genomic DNA libraries and complementary DNA (cDNA) libraries -

Genomic Library

-consists of many overlapping fragments of genomes with at least one copy of every DNA sequence in an organism's genome -made by extracting DNA from cells or tissues and cut with restriction enzymes -resulting fragments are inserted into vectors -contain coding and noncoding segments of DNA such as introns

Complementary DNA (cDNA) Libraries

-contain complementary DNA copies or cDNA made from the mRNAs present in a cell population -represent the genes being expressed in the cells at the time the library was made -contain expressed genes -can be used to compare expressed genes from normal tissues and diseased tissues -valuable in identifying genes involved in cancer formation -prepared by: >>>isolating mRNA from cells >>>sythesizing complementary DNA using reverse transcriptase >>>cloning cDNA molecules into a vector -reverse transcriptase polymerase chain reaction (RT-PCR) can be used to generate cDNA from mRNA by: >>>first making a single-stranded cDNA copy of the mRNAs using reverse transcriptase >>>then using PCR to copy the single-stranded DNA into double-stranded DNA -provide an instant catalog of all genes active in a cell at a specific time -very valuable tools for scientists isolating and studying genes in a particular tissue

Bacterial Plasmid Vectors

-plasmid: extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells/ accessory DNA found in some bacterial cells -genetically modified were the first vectors developed -can be commercially prepared and readily available with a range of useful features -can make specific genes/DNA sequences to follow characteristics of plasmids -used for DNA cloning usually have been engineered to contain: >>>number of convenient restriction sites >>>marker gene to select for its presence in host cell -introduced into bacteria by the process of transformation, which is achieved through two processes 1. Using calcium ions and brief heat shock to pulse DNA into cells 2. Electoporation uses a brief but high-intensity pulse of electricity to move DNA into bacterial cells -only a few enter a bacterial host through transformation, but because they have their own origin of replication or ori, they can produce several hundred copies in a single host -have also been genetically engineered to contain a number of restriction sites vs. only one naturally occuring in bacteria >>>for commonly used restriction enzyme in a region called multiple cloning site >>>allows scientists to clone a range of different fragments generated by many commonly used restriction enzymes -Both plasmid DNA and DNA to be cloned are cut with same restriction enzyme -cut once within multiple cloning site to produce a linear vector >>>DNA restriction fragments from DNA to be cloned are added to linearized vector in presence of DNA ligase >>>Sticky ends of DNA fragments anneal, joining the DNA to be cloned with the plasmid >>>recombinant DNA is produced and is the introduced into bacterial host cells by transformation -only one or a few plasmids enter a host cell -once inside cell, plasmids replicate quickly to produce multiple copies -bacterial cells containing recombinant DNA can be readily identified by using selectable marker genes -Genes that provide resistance to antibiotics such as ampicillin and genes such as lacZ are very effective selectable markers -transformed bacteria plated on agar plates that contain an antibiotic such as ampicillin form white colonies and are clones of each other, genetically identical cells with copies of recombinant plasmids -nontransformed bacteria cannot grow well on these plates because they do not have ampicillin resistance gene: few that grow form blue colonies because they produce lacZ enzyme to metabolize X-gal -lacZ gene in plasmid is disrupted due to cloning and transformed cells cannot metabolize X-gal and therefore form white colonies

Polymerase Chain Reaction (PCR)

-powerful technique for copying DNA -rapid method of cloning DNA, eliminates need to use host cells for cloning -amplifies DNA sequences present in very small quantities -double-stranded DNA to be cloned is put in a tube with DNA polymerase Mg2+ and the four deoxyribonucleoside triphosphates -requires two olgionucleotide primers (short stranded sequences) >>>one complementary to the 5' end of one strand of target DNA to be amplified >>>other complementary to the 3' end of other strand -primers bind to denatured DNA and complementary strands are synthesized by a heat-stable DNA polymerase -Taq Polymerase is thermostable, was isolated from Thermus aquaticus -three steps repeated over and over using a thermocycler to amplify the DNA exponentially: 1. Denaturation 2. Primer Annealing 3. Extension -DNA strand is doubled in each cycle, and news strands along with old strand serve as templates in next cycle -rapid, carried out in a few hours -very sensitive, amplifying sequences from very small DNA samples

Nucleic Acid Blotting

-process of transferring proteins, DNA, or RNA onto a carrier such as nitrocellulose or nylon -allows for hybridization between complementary nucleic acid (DNA or RNA) molecules -Southern Blot -Northern Blot -Western Blot

Restriction Enzymes

-recognize and bind to DNA at a specific nucleotide sequence or restriction site -cleave DNA to produce restriction fragments -cut DNA at specific sites -restriction sites are present randomly in the genome and are mostly four to six nucleotides long but some contain eight or more -most recognition sequences exhibit a form of symmetry described as a palindrome >>>nucelotide sequence reads the same on both strands in 5' to 3' direction -each recognizes its particular recognition site and cuts the DNA in a characteristic cleavage -some enzymes make offset cuts producing fragments with single overhanging ends called cohesive or sticky ends, while others make cuts on both ends in the same nucleotide to produce blunt-end fragments -When DNA fragments produced by the same restriction enzymes are mixed together, fragments can anneal or stick together by hydrogen bonding of complementary base pairs in their single-stranded ends -DNA ligase seals the phosphodiester backbone covalently to produce intact DNA molecules

Whole Genome Shotgun Cloning and New Sequencing Methodologies/Next-Generation Sequencing

-replacing traditional genomic libraries -allow one to sequence genomic DNA fragments without inserting DNA fragments into vectors and cloning them in host cells

Oligo-

-short

Limitations of PCR

-some information about the nucleotide sequence of target DNA must be known in order to synthesize primers -minor contamination of sample DNA from other sources can cause problems -cannot amplify long segments of DNA, only those that are relatively short >>>several thousand nucleotides in length

Southern Blot

-used to identify hybridization between complementary DNA molecules -used to identify which clones in a library contain a given DNA sequence -used to characterize the size of the fragments -used to identify fragments carrying specific genes in genomic DNA digested with restriction enzymes -used to determine whether a clone contains all or part of a gene -used to ascertain the size and sequence of organization of a gene or DNA sequence of interest -used in forensic applications -two components: 1. Separation of DNA fragments by Gel Electrophoresis 2. Hybridization by using labeled probes -DNA is often cut into fragments with one or more restriction enzymes -Fragments are separated by Gel Electrophoresis -For hybridization: >>>DNA in gel is denatured to form single strands >>>gel is overlaid with DNA-binding membrane or nylon >>>DNA fragments are transferred to membrane >>>membrane is placed on heat-sealed bag with a labeled, single-stranded DNA probe >>>excess probe is washed away and hybridized fragments are visualized on a piece of film

Probes

-used to screen a library to recover clones of a specific gene -any DNA or RNA sequence that is complementary to the target gene of sequence to be identified -use cloned genes from rats, mice, or Drosophilia with conserved sequence similar to human genes to identify human genes -to screen a library, bacterial clones from library are grown on nutrient agar plates to produce colonies -colonies are screened by transferring bacterial colonies from the plate to a filter and hybridizing the filter with a nucleic acid probe to the DNA sequence of interest -colony corresponding to the one the probe identified on the filter is identified and recovered

Library Screening

-used to sort through a library and isolate specific genes of interest

Reverse Transcription PCR (RT-PCR)

-used to study gene expression by examining mRNA production by cells or tissues

Bacterial Artificial Chromosome (BACs)

-vectors that can be used to clone large fragments of DNA -generally very large but are low copy number plasmids (one or two copies per bacterial cell) -accept DNA inserts of sizes 100-300 kb

Yeast Artificial Chromosomes (YACs)

-vectors that can be used to clone large fragments of DNA -have telomeres at each end, origins of replication, and a centromere -range in size from 230 kb-1900+kb -important tool in human genome project -modified and available commercially -commonly used to accommodate large sizes of DNA necessary to span the 3 billion bp of DNA in the human genome

Applications of PCR

-very useful tool since it allows the screening of mutations involved in genetic disorders -a key diagnostic methodology for detection of bacteria and viruses in humans and pathogenic bacteria such as E. Coli and Staphylococcus aureus in contaminated food -techniques are particularly advantageous when studying samples from single cells, fossils, or a crime scene where a single hair or even saliva-moistened postage stamp is the source of DNA -enforce worldwide ban on sale of certain whale products -determine pedigree background of purebred dogs

Ways Genes are Introduced to Plants

-widely used approach: inserting genes into plant cells using Rhizobium radiobacter >>>infects plant cells >>>produces tumors or crown galls in many plant species >>>contains a plasmid called Ti plasmid >>>restirction sites in Ti plasmids are used to make recombinant vectors >>>cells are transformed with the recombinant vectors >>>that containing recombinant DNA is mixed w/ plant cells >>>once inside cell, foreign DNA is inserted into host cell chromosome by Ti plasmid and grow to produce a mature plant

Western Blot

-widely used to analyze proteins -used to determine whether a gene is actively being expressed in a given cell or tissue -used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders

Recombinant DNA Technology Began with Two Key Tools

1. Restriction Enzymes/DNA-Cutting Enzymes 2. DNA Cloning Vectors

Gel Electrophoresis

=separates fragments by size -smallest fragments move farthest in gel and can be visualized when stained with ethidium bromide and illuminated by UV light -cutting pattern of fragments generated is interpreted to determine the location of restriction sites -bars represent individual fragments of DNA