CHAPTER 3 Recombinant DNA Technology and Genomics Power. Point® Lecture by: Melissa Rowland-Goldsmith Chapman University
Chapter Contents • 3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • 3. 2 What Makes a Good Vector? • 3. 3 How Do You Identify and Clone a Gene of Interest? • 3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • 3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • 1970 s: Gene cloning became a reality – Clone – a molecule, cell, or organism that was produced from another single entity • Made possible by the discovery of: – Restriction Enzymes – DNA cutting enzymes (molecular scissors) – Plasmid DNA Vectors – circular form of self-replicating DNA • Can be manipulated to carry and clone other pieces of DNA © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Restriction Enzymes Primarily found in bacteria (they use these for defense) – Cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotides in a DNA strand – Bind to, recognize, and cut DNA within specific sequences of bases called a restriction site • Each restriction site is a palindrome – reads same forward and backward on opposite strands of DNA – There are 4 or 6 bp cutters because they recognize restriction sites with a sequence of 4 or 6 nucleotides © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Why don't restriction enzymes digest bacteria DNA? © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Restriction enzymes a. Some cut DNA to create DNA fragments with overhanging single stranded ends called "sticky" or "cohesive" ends b. Some cut DNA to generate fragments with double-stranded ends called "blunt" ends http: //highered. mheducation. com/sites/0072 556781/student_view 0/chapter 14/animation_ quiz_6. html © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Would Eco. RI cut the following sequence? Work in groups to explain your answer. • 5'CTCGAGTTCGAG 3' • 3'GAGCTCAAGCTC 5' © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Restriction enzymes • Advantage of enzymes that produce sticky ends – Preferred for cloning because DNA fragments with sticky ends can be easily joined together because they base pair with each other by forming weak hydrogen bonds – Work in groups and use Table 3. 1 to choose two enzymes you would use for cloning and then state your reasons. © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Plasmid DNA – small circular pieces of DNA found primarily in bacteria • Are considered extrachromosomal DNA because they are in the cytoplasm in addition to the bacteria chromosome • Are small approximately 1 to 4 kb • Can replicate independently of chromosome • Can be used as vectors – pieces of DNA that can accept, carry, and replicate other pieces of DNA © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Creating recombinant DNA • Look at Table 3. 1, if you used the restriction enzyme Sma. I for both the insert and plasmid, would there be hydrogen bonds of the blunt ends? © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Recombinant DNA • 1975 NIH formed the RAC (Recombinant DNA Advisory Committee) based on results from Asilomar meeting – Purpose of RAC: evaluate recomb. technology and establish guidelines for research – 1976 RAC published set of guidelines for working with recombinant organisms • Work in groups to discuss one situation that could be harmful to humans if RAC did not exist © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Transformation of Bacterial Cells – very inefficient process – A process for inserting foreign DNA into bacteria • • Treat bacterial cells with calcium chloride Add plasmid DNA to cells chilled on ice Heat the cell and DNA mixture Plasmid DNA enters bacterial cells and is replicated and express their genes – electroporation – Apply brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall that allow the DNA to enter © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Selection of recombinant bacteria after transformation – Selection is a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria and bacteria that contain plasmid without foreign DNA 1. Antibiotic selection – plate transformed cells on plates containing different antibiotics to identify recombinant bacteria and non-transformed bacteria – Does not select for plasmid containing foreign DNA vs. recircularized plasmid © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Selection of recombinant bacteria after transformation 2. Blue-white selection – DNA is cloned into the restriction site in the lac. Z gene – When it is interrupted by an inserted gene, the lac. Z gene cannot produce functional Beta gal – When Xgal (artificial lactose) is added to the plate, if functional lac. Z is present = blue colony – Non-functional lac. Z = white colony = clone = genetically identical bacterial cells each containing copies of recombinant plasmid © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning © 2013 Pearson Education, Inc.
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Assume you used a plasmid that contains the lacz gene in the restriction enzyme site. The plasmid has an antibiotic resistance gene. Following transformation, you grow up the cells on an agar plate containing the antibiotic. Here are your results. • Work in groups of two and discuss which colonies have the inserted gene. 1 2 3 5 © 2013 Pearson Education, Inc. 4 6
3. 1 Introduction to Recombinant DNA Technology and DNA Cloning • Introduction to human gene cloning • First human protein expressed via recombinant techniques was insulin and next was growth hormone • Clone human insulin DNA sequence into a plasmid and the bacteria cells were then used to synthesize the protein product of the cloned gene • Can generate lots of pure protein via this technique • What was source of growth hormone prior to recombinant technology? © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Practical Features of DNA Cloning Vectors – Size – small enough to be separated from chromosomal DNA of host plasmid – Origin of replication (ori) – site for DNA replication that allow plasmids to replicate independently from host chromosome • Copy number: number of plasmids in the cell (normally small but plasmids have high copy numbers) – Multiple cloning site (MCS) – recognition sites for several restriction enzymes in which insert is cloned into – Selectable marker genes – allow to select for transformed colonies – RNA polymerase promoter sequences – used for transcription in vitro and in vivo – DNA sequencing primers © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Types of Vectors – Bacterial plasmid vectors – can clone inserts that are smaller than 7 kb; some express eukaryotic proteins from genes poorly – Bacteriophage vectors – Cosmid vectors – Expression vectors – Bacterial Artificial Chromosomes (BAC) – Yeast Artificial Chromosomes (YAC) – Ti vectors © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Types of vectors – Bacteriophage vectors – advantage: clone up to 25 kb λ genome is linear and 49 kb – Cloned DNA is inserted into restriction sites in center of λ chromosome – Recomb. chromosomes are packaged into viral particles in vitro – These phages then infect lawn of E coli cells – At each end of λ are 12 bp sites = COS which base pair together when they infect bacteria and circularize and replicate – Obtain plaques – zones of dead bacteria which contain millions of recombinant phage particles © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Cosmid vectors: contain • • • COS ends of λ DNA Plasmid ori of rep Gene for antibiotic resistance DNA is cloned into restriction site Then cosmid is packaged into viral particles and used to infect E coli cells at low copy number • Bacterial colonies are grown on the plate and recombinants are screened by antibiotic selection Advantages: clone fragments between 20– 40 kb • Discuss the cosmid features shared by either a plasmid and bacteriophage © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Expression vectors – Bacteria expression vector: • Allow high level protein expression in bacterial cells because they have a prokaryotic promoter site next to the MCS • Bacterial RNA POL can then bind to promoter and transcribe the insert's sequence which is then translated into protein • Protein is then purified using biochem. techniques • Problems with bacteria expression vectors: sometimes bacteria ribosomes cannot translate eukaryote sequence or protein is not folded correctly since bacteria does not have organelles for processing. It is not possible to use this system with eukaryote genomic DNA. © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Assume you want to make lots of human insulin using a bacteria expression vector. • Work in groups of two and discuss why using human insulin genomic DNA for this cloning project would not be advantageous. © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Bacteria artificial chromosomes (BAC) – Large low copy plasmids – Contain genes that encode the F factor (unit of genes controlling bacterial replication) – Can accept large sizes of DNA inserts ranging from 100– 300 kb – Were used during the human genome project to clone and sequence large pieces of chromosomes © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Yeast artificial chromosomes (YAC) – Is miniature version of eukaryotic chromosome containing ori or rep; two telomeres; selectable markers; centromere that allows replication of YAC and segregation of daughter cells – Best for cloning very large DNA inserts from 200 kb to 2 megabases – Were used for human genome project – Small plasmids grown in E coli and introduced to yeast cells (S. cervisiae) © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? • Ti vector – Naturally occurring plasmids isolated from the bacterium that is a soil plant pathogen causing disease in plants – When the bacteria infects plant cells, the T DNA from the Ti plasmid inserts into the host chromosome – T DNA codes for auxin hormone that weakens plant cell wall and infected plants divided and enlarge to form a tumor (gall) – Scientists use Ti vectors to deliver genes to plants by removing toxic gene for auxin © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Creating DNA Libraries – Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host – Screened to pick out different genes of interest • Two Types of Libraries – Genomic DNA libraries – Complementary DNA libraries (c. DNA libraries) © 2013 Pearson Education, Inc.
3. 2 What Makes a Good Vector? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Genomic Libraries – Chromosomal DNA from the tissue of interest is isolated and digested with a restriction enzyme which produces many fragments that include the entire genome – Vector is digested with same enzyme – DNA ligase is used to ligate genomic DNA fragments and vector DNA – Recombinant vectors are used to transform bacteria and theoretically each bacteria will contain a recombinant plasmid © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? – Disadvantages of genomic libraries • Introns are cloned in addition to exons; – Majority of genomic DNA is introns in eukaryotes so majority of the library will contain non-coding pieces of DNA • Many organisms have very large genome, so searching for gene of interest is difficult • Time consuming! © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • c. DNA Libraries – m. RNA from tissue of interest is isolated – Need to make double stranded DNA from m. RNA: How? a. enzyme reverse transcriptase catalyzes synthesis of complementary single stranded DNA from m. RNA i. Called complementary DNA (c. DNA) because it is an exact copy of the m. RNA b. m. RNA is degraded either with an enzyme or alklaline solution c. DNA Pol is used to synthesize second strand of DNA to create double stranded c. DNA – Short linker double stranded DNA sequences which contain restriction enzyme recognition sites are added to the ends of the c. DNA – Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors – Then transform bacteria with recombinant vectors © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • c. DNA Libraries – Advantage over genomic libraries • Collection of actively expressed genes in the cells or tissues from which the m. RNA was isolated • Introns are NOT cloned • Can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue • Assume that a gene involved in increased muscle mass is expressed when the muscle cells are exposed to growth hormone. What would be the source of the c. DNA library: muscle cells or muscle exposed to growth hormone? Work in groups to explain your answer. – Disadvantage • Can be difficult to make the c. DNA library if a source tissue with an abundant amount of m. RNA for the gene is not available © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Library screening to identify the gene of interest • Colony hybridization – Bacterial colonies containing recombinant DNA are grown on an agar plate – Nylon or nitrocellulose filter is placed over the plate and some of the bacterial colonies stick to the filter at the exact location they were on the plate – Treat filter with alkaline solution to lyse the cells and denature the DNA – Denatured DNA binds to filter as single-stranded DNA – Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye • DNA fragment that is complementary to the gene of interest – Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Why is it necessary to tag the probe with either a radioactive nucleotide or fluorescent dye that can be used to catalyze light releasing reactions? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Colony Hybridization – Filter is washed to remove excess unbound probe – Filter is exposed to film – autoradiography • Anywhere probe has bound to the filter, radioactivity from the radioactive probe or released light (fluorescence or chemiluminescence) from non-radioactive probes exposes silver grains in the film – Depending on the abundance of the gene of interest there might be few colonies or plaques on the filter that hybridize to the probe • Film is developed to create a permanent record of the colony hybridization – Use digital instrument to detect probe binding if a fluorescent or chemiluminescent probe was used – Film is then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Colony Hybridization – Type of probe used depends on what is already known about the gene of interest – Example: use mouse or rat probe to screen a human library because many genes between these species are similar – If gene sequence has NOT been cloned in another species but something is known about the protein, what can be done? • Library screening rarely results in the cloning of the fulllength gene – Usually get small pieces of the gene; the pieces are sequenced and scientists look for overlapping sequences – Look for start and stop codons to know when the full length of the gene is obtained © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Polymerase Chain Reaction – Developed in the mid-1980 s by Kary Mullis – Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time – Process • Target DNA to be amplified is added to a tube, mixed with nucleotides (d. ATP, d. CTP, d. GTP, d. TTP), buffer, and DNA polymerase. • Paired set of Forward and Reverse Primers are added – short single-stranded DNA oligonucleotides (20– 30 bp long) – Primers are complementary to nucleotides flanking opposite ends of target DNA • Reaction tube is placed in an instrument called a thermocycler © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • PCR Process continued – Thermocycler will take DNA through a series of reactions called a PCR cycle – Each cycle consists of three stages 1. Denaturation – heat to 94 °C to 96 °C 2. Annealing (hybridization) – in which primers H bond with complementary bases at the opposite ends of target sequence at 55 °C to 65 °C 3. Extension (elongation) – DNA Pol copies target DNA at 70 to 75 °C – At the end of one cycle, the amount of DNA has doubled – Cycles are repeated 20– 30 times © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Advantage of PCR: • Can amplify millions of copies of target DNA from small amount of starting material in short period of time • To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation 2 N in which N represents number of PCR cycles • Assume you want to do 22 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • The type of DNA polymerase used is very important – Taq DNA polymerase – isolated from a species known as Thermus aquaticus that thrives in hot springs – Why can't you use DNA Pol isolated from bacteria that live at 37 C? • Applications – – – Making DNA probes Studying gene expression Detection of viral and bacterial infections Diagnosis of genetic conditions Detection of trace amounts of DNA from tissue found at crime scene – Detection of DNA from fossilized dinosaur tissue © 2013 Pearson Education, Inc.
How Do You Identify and Clone a Gene of Interest? © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? • Cloning PCR Products – Is rapid and effective compared to using DNA libraries – Disadvantage • Need to know something about the DNA sequence that flanks the gene of interest to design primers • Assume the human genome project was not completed but you wanted to clone growth hormone from humans, what sequence would you use to design PCR primers? – Great trick: Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products • Use this knowledge to researcher's advantage: T vector that has single stranded thymine on each end so can complementary base pair with the adenine in the PCR products © 2013 Pearson Education, Inc.
3. 3 How Do You Identify and Clone a Gene of Interest? © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Agarose Gel Electrophoresis: separate and visualize DNA fragments based on size • Agarose is isolated from seaweed and when melted in a buffer solution and poured into a horizontal tray and as it cools it will form a semisolid gel containing small pores through which DNA will travel • The percentage of agarose used to make the gel determines the ability of the gel to separate DNA fragments of different sizes • (gel % range from 0. 5 to 2%) • High % gel (2%) allows to resolve smaller size fragments • Low % (0. 5%) resolves larger size fragments • What size fragments would be resolved using a 1% gel? © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Agarose Gel Electrophoresis – To run a gel, it is submerged in a buffer solution that conducts electricity – DNA is loaded into small depressions called wells at the top of the gel – Electric current is applied through electrodes at opposite ends of the gel • DNA migrates according to its charge and size • Rate of migration through the gel depends on the size of the DNA because the sugar phosphate backbone makes it always negatively charged • DNA migrates toward positive pole and is repelled by negative pole © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Agarose Gel Electrophoresis • Migration distance is inversely proportional to size of DNA fragment – Large fragments migrate slowly; smaller fragments migrate faster – Tracking dye is added to the samples to monitor DNA migration during electrophoresis – DNA can be visualized after electrophoresis by the addition of DNA staining dyes • Ethidium bromide: intercalate between DNA base pairs and it fluoresces under ultraviolet light • Then a picture can be taken to document the gel results © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology M Digest basepairs 2000 1500 1000 A B C 500 • Here is a result in which you ran your DNA fragments on a 1% gel. What is the approximate size of band B? Why did it not migrate super fast through the gel? © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Restriction mapping gene structure – Cut cloned gene with restriction enzymes to pinpoint location of the cutting sites – Knowing restriction map is useful for making clones of small pieces of the DNA which is called subcloning – These small pieces of DNA can then be sequenced – Protocol of restriction mapping: a. Digest DNA with single or double restriction enzymes b. Separate DNA fragments via agarose gel electrophoresis c. Arrange fragments in order to make map of restriction sites © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology *** Note: These days scientists use bioinformatic software to identify restriction sites in the DNA © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • DNA Sequencing – Important to determine the sequence of nucleotides of the cloned gene – Work in groups of two to discuss at least three reasons why it is important to know the sequence of the cloned DNA – Chain termination sequencing (Sanger method) – Requires single stranded primer annealing to denatured DNA template and then the reaction tube also contains all 4 d. NTPs; DNA Pol, and dideoxynucleotide (dd. NTP) which has a 3' H instead of 3'OH on the deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide and so gets terminated © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Original Sanger method: – Had four separate reaction tubes and each contained the vector; primer; d. NTPs in which one d. NTP is radioactively labeled; and a different small amount of dd. NTP; DNA Pol – Over time a dd. NTP will be incorporated into all the positions in the newly synthesized strands creating fragments of varying lengths that are terminated at the dd. NTP – Then the fragments are separated on polyacryamide gel – Autoradiography used to then identify radioactive fragments – Read the gel from bottom to top as individual nucleotides – The sequence generated from the reaction is complimentary to the sequence on the template strand in the vector © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Read the first 24 nucleotide sequence from the gel generated in Figure 3. 12 © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology – High throughput computer automated sequencing using Sanger method- instead of sequencing 200– 400 nucleotides per reaction this allowed greater than 500 nucleotides to be sequenced per reaction – Was very helpful for completing human genome project – Procedure: instead of 4 separate tubes, only 1 reaction tube is used • dd. NTPs are each labeled with a different fluorescent dye • Samples are separated on a single-lane capillary gel that is scanned with a laser beam • Laser stimulates fluorescent dye on each DNA fragment which emits a different wavelength of light for each different colored dd. NTP • Emitted light is collected by a detector that amplifies and feeds this info. to a computer that can run multiple capillary gels at one time = 900 bp sequence • Computer converts light patterns to reveal the sequence © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • • Next generation sequencing (NGS) = designed to produce highly accurate and long stretches of DNA sequence (greater than 1 gigabase) of DNA per reaction Is done at very low cost Use parallel formats that use fluorescence imaging techniques Two types of NGS approaches A. Roche 454 commercial system using pyrosequencing: – Pyrosequencing: beads are attached to fragmented genomic DNA which is PCR amplified in separate water droplets in oil for each bead then loaded into multiwell plates and mixed with DNA POL; then actual pyrosequencing – Pyrosequencing: single d. NTP is flowed over the wells and if it incorporated into complementary sequencing strand, a pyrophosphate is released due to making a phopshodiester bond and then a series of chemiluminescent (light releasing) reactions take place to ultimately produce light with firefly luciferase enzyme • Emitted light from the reaction is captured and recorded to determine when a single nucleotide has been added to the growing strand • By rapidly repeating the nucleotide flow step with each of the 4 d. NTPs, can obtain reads of approx. 400 nucleotides and order of 400 million bases of data per 10 hour run © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • The other type of NGS approach: B. ABI SOLID (supported oligonucleotide ligation and detection) method – Can produce 6 gigabases of sequence data per run – Similar to 454 approach in which DNA fragments are linked to beads and amplified but different sequencing technologies used C. Future = third generation sequencing using nanotechnology to push single strand nucleotide fragments into nanopores and then cleaving off individual bases to produce a signal that can be captured – This new approach will not involve DNA amplification or fluorescent tags so it is truly direct sequencing © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Fluorescence in situ hybridization (FISH) • Chromosome Location and Copy Number – Identify which chromosome contains a gene of interest – Procedure: • Chromosomes are isolated from cells and spread out on glass microscope slide • DNA or RNA probe for gene of interest is labeled with fluorescent nucleotides and incubated with slides • Probe will hybridize with complementary sequences on chromosomes on slide • Slide is washed and exposed to fluorescent light • Wherever probe has bound to the chromosome, it is illuminated to indicate the presence of the probe binding • Do karyotype to determine which chromosome shows fluorescence © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology – Fluorescence in situ hybridization (FISH) continued • To determine which chromosome shows fluorescence they are aligned according to their length and staining patterns of their chromatids to create a karyotype • What does fluorescence on more than one chromosome indicate? • FISH used to analyze genetic disorders • FISH used to determine which cells in a particular organ are expressing the particular m. RNA © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology – Southern blotting – Used to determine gene copy number; gene mapping; gene mutation detection; PCR product confirmation; DNA fingerprinting • Digest chromosomal DNA into small fragments with restriction enzymes • Fragments are separated by agarose gel electrophoresis • Gel is treated with alkaline solution to denature the DNA • Fragments are transferred onto a nylon or nitrocellulose filter (called blotting) • Filter (blot) is baked or exposed to UV light to permanently attach the DNA • Filter (blot) is incubated with a labeled probe and exposed to film by autoradiography • Number of bands on film represents gene copy number © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression – Techniques involve analyzing m. RNA produced by a tissue – Northern blot analysis • Basic method is similar to Southern blotting • RNA is isolated from a tissue of interest, separated by gel electrophoresis, blotted onto a membrane, and hybridized to a labeled DNA probe, exposed bands on autoradiograph show presence of m. RNA for gene of interest as well as size of m. RNA • Can compare and quantify amounts of m. RNA present in different tissues © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression – Techniques involve analyzing m. RNA produced by a tissue – Northern blot analysis © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression • Reverse transcription PCR- used to study m. RNA levels when level of detection is below that of Northern • Procedure: • Isolate m. RNA and use Reverse Transcriptase to make double stranded c. DNA • What procedure did you already learn about using RT in this chapter? • Use PCR to amplify region of c. DNA with set of primers specific for gene of interest • Run agarose gel to separate amplified fragments • Determine expression patterns in the tissue • **Amount of c. DNA produced in RT PCR rxn for gene of interest reflects amount of m. RNA and level of gene expression © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression • Real time or quantitative (q. PCR) • Can quantify amplification reactions as they occur in real time • Need special thermal cyclers that use a laser to scan a beam of light through the top or bottom of each PCR reaction • Each reaction tube contains EITHER a dye containing probe or DNA binding dye that emits fluorescent light when illuminated by the laser • Light emitted by the dyes correlates with amount of PCR product amplified • Light is captured by the detector which relays info. to the computer to provide readout on amount of fluorescence • Readout is plotted analyzed to quantify the number of PCR products produced after each cycle © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression • • Real time or quantitative (q. PCR) Two approaches a. Taqman probes are complimentary to specific regions of target DNA between forward and reverse primers for PCR – Taqman probes contain two dyes: reporter located at 5' end of probe and can release fluorescent light when excited by the laser and other dye is quencher which is attached to 3' end of probe (see figure for more details) B. SYBR green- binds double stranded DNA and as more double stranded DNA is copied with each round of q. PCR there are more DNA copies to bind SYBR Green which increases amount of fluorescent light emitted © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression – Gene microarrays enable researchers to study all of the genes expressed in a tissue very fast – Microarray (gene chip) is created with use of small glass microscope slide • Single stranded DNA molecules are spotted on the slide using an arrayer (computer controlled robotic arm) which fixes DNA (multiple copies of c. DNA) at different spots on the slide which is recorded by a computer © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression – Gene microarrays • Procedure to do array: – Extract m. RNA from a tissue of interest and c. DNA is synthesized from m. RNA and labeled with fluorescent dye – Labeled c. DNA is incubated overnight with the array where it hybridizes with different spot on the array that contain complementary DNA sequences – Can have over 10, 000 spots of DNA – Array is washed and scanned by a laser that causes c. DNA hybridized to array to fluoresce – Fluorescent spots reveal which genes were regulated and Intensity of fluorescence indicates relative amount of gene expression © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology • Studying Gene Expression – Gene microarrays – Common to do microarray in which you compare two different conditions with 2 different colored dyes (one for treatment and other for control condition) – Laser is scanned at different wavelengths for each probe and then the images are overlaid to make direct comparisons between the treatment and control • Example study gene expression difference between cancer cells and normal cells to look for genes possibly involved in cancer progression • Results of such studies can possibly lead to new drug therapies to combat cancer and other diseases © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology – Gene mutagenesis studies • To study structure and function of protein produced by a specific gene • Site directed mutagenesis: mutations created in specific nucleotides of a cloned gene contained in a vector • Gene is then expressed in cells which results in translation of a mutated protein • This procedure lets researchers study effects of mutation on protein structure as well as provides information about which nucleotides are important for specific functions of a protein • Very useful in helping identify critical sequences in a gene that produced proteins involved in disease © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology – RNA interference (RNAi) = naturally occurring mechanism for inhibiting gene expression Procedure: – Doublestranded RNA can be bound by Dicer enzyme that cuts ds. RNA into 21– 25 nucleotide snippets called small interfering RNA (si. RNA) – si. RNA then bound to protein: RNA complex called RNA inducins silencing complex (RISC) – RISC unwinds ds. RNA releasingle stranded RNA that bind complementary m. RNA – Binding of si. RNA to m. RNA leads to degradation of m. RNA by Slicer enzyme or blocks translation by interfering with ribosome binding – Currently developing techniques to use RNAi to silence gene expression © 2013 Pearson Education, Inc.
3. 4 Laboratory Techniques and Applications of Recombinant DNA Technology © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • Genomics – cloning, sequencing, and analyzing entire genomes – Whole genome shotgun sequencing or shotgun cloning • Use restriction enzymes to digest pieces of entire chromosomes: – Produces thousands of overlapping fragments called contigs which are each sequenced. – Computer programs are used to align the sequenced fragments based on overlapping sequence pieces © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • Bioinformatics: Merging molecular biology with computer technology – An interdisciplinary field that applies computer science and information technology to promote an understanding of biological processes • Application of Bioinformatics – Databases to store, share, and obtain the maximum amount of information related to gene structure, gene sequence and expression, and protein structure and function © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology – Gen. Bank = public database of DNA sequences and contains National Institute of Health collection of DNA sequences • Each entry has an accession number that scientists use to refer back to the cloned sequence • Maintained by the National Center for Biotechnology Information (NCBI) – NCBI is goldmine for bioinformatics resources that creates public access databases and develops computing tools for analyzing and sharing genome data © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology – DNA database searching • Basic Local Alignment Search Tool (BLAST) – Used to search Gen. Bank for sequence matches between cloned genes and to create DNA sequence alignments © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • The Human Genome Project – Started in 1990 by the U. S. Department of Energy – International collaborative effort to identify all human genes and to sequence all the base pairs of the 24 human chromosomes – Mostly done by 20 centers in 18 countries: China, France, Germany, Great Britain, Japan, and the United States – Competitor was a private company, Celera Genomics, directed by Dr. J. Craig Venter © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • The Human Genome Project designed to accomplish the following: • Analyze genetic variations among humans. This included the identification of single-nucleotide polymorphisms • Map and sequence the genomes of model organisms, including bacteria, yeast, roundworms, fruit flies, mice, and others • Develop new laboratory technologies such as high-powered automated sequencers and computing technologies, as well as widely available databases of genome information, which can be used to advance our analysis and understanding of gene structure and function • Disseminate genome information among scientists and the general public • Consider the ethical, legal, and social issues that accompany the HGP and genetic research © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • The Human Genome Project – April 14, 2003: map of the human genome was completed – Consists of 20, 000 protein-coding genes – Map was complete with virtually all bases identified and placed in their order and potential genes assigned to chromosomes • Why are there only 20, 000 genes coding for proteins when it was predicted that there would be 100, 000 genes? Work in pairs to answer this question. © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • What we have learned from HGP – Nearly 50% genes do not yet have a function • Summary of findings: – The human genome consists of approximately 3. 1 billion base pairs – The genome is approximately 99. 9% the same between individuals of all nationalities – Single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs)—such as long deletions, insertions and duplications in the genome—account for much of the genome diversity identified between humans – Less than 2% of the genome codes for genes – The vast majority of our DNA is non-protein coding, and repetitive DNA sequences account for at least 50% of the noncoding DNA – The genome contains approximately 20, 000 protein-coding genes © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • • • What we have learned from HGP Many human genes are capable of making more than one protein, allowing human cells to make at least 100, 000 proteins from only about 20, 000 genes. Chromosome 1 contains the highest number of genes. The Y chromosome contains the fewest genes. Many of the genes in the human genome show a high degree of sequence similarity to genes in other organisms. Thousands of human disease genes have been identified and mapped to their chromosomal locations © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • The Human Genome Project – Started an "omics" revolution • Proteomics—studying all proteins in a cell • Metabolomics—studying proteins and enzymatic pathways involved in cell metabolism • Glycomics—studying carbohydrates of a cell • Transcriptomics—studying all genes transcribed in a cell • Metagenomics—analysis of genomes of organisms in an environment • Pharmacogenomics—customized medicine based on person's genetic profile for a particular condition • Nutrigenomics—interaction between genes and diet © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • Comparative Genomics – Mapping and sequencing genomes from a number of model organisms – Allows researchers to study gene structure and function in these organisms in ways designed to understand gene structure and function in other species including humans © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • Stone Age Genomics (paleogenomics) – Analyzing "ancient" DNA © 2013 Pearson Education, Inc.
3. 5 Genomics and Bioinformatics: Hot Disciplines of Biotechnology • 10 years after completion of HGP—what next? – Human epigenome project—create maps of epigenetic changes in different cell and tissue types – International Hapmap Project—characterization of SNPs for their role in genome variation, disease and pharacogenomics applications – Encyclopedia of DNA elements (ENCODE)—use experimental approaches and bioinformatics to ID and analyze functional elements that regulate expression of human genes – Personalized genome projects – Cancer genome project—map important genes and genetic changes involved in cancer © 2013 Pearson Education, Inc.
Скачать презентацию CHAPTER 3 Recombinant DNA Technology and Genomics Power
b63aa62ed35997479fbca7657dd6bbf8.ppt