Biotechnology. terms. terms. Exam 2 In two weeks! Tuesday Nov. 3 rd. Genetic Engineering & molecular techniques

Transcription

1 Molecular Genetics and Genetic Engineering Review sessions wt Jackie: 3-5PM Thursdays, room 8 Illick Previous: Exam 2 In two weeks! Tuesday Nov. 3 rd Biotechnology birth of modern biotechnology Recombinant human insulin first produced & began widely use in 5 years % US soybean, 76% US cotton, & 45% US corn is transgenic Next two lectures together: Genetic Engineering adds just a few genes to the genome Genetic Engineering & molecular techniques This section: Basics of GE Basic molecular techniques GE of plants GE of animals Discussion terms Recombinant DNA: DNA resulting from combining DNA from two sources Examples Crossing over during meiosis to increase genetic diversity Chromosomal rearrangements which can help drive evolution (Ex. Gene duplication) Transposons and some viruses Development of immune response cells Modern molecular techniques In a test tube mimic what can happen in nature terms Cloning: making genetically identical (or nearly identical) copies of DNA molecules, cells, or whole organisms Examples Bacterial replication produce clones Identical twins are clones Plants propagated from cuttings are clones 1

2 terms Making a recombinant DNA clone Genetic engineering: using the processes of cloning and recombinant DNA to mindfully modify a gene, cell, or organism Started in early 1970 s Technology older than most of you Basics for making recombinant DNA (rdna) Plasmids Vectors to carry rdna Restriction endonucleases To cut DNA DNA ligase To connect DNA E. coli Used to amplify cloned DNA Plasmids Bacterial extra-chromosomal DNA Circular DNA molecules Contain an origin of replication Stringent: 1-2 copies per cell Relaxed: usually over 100 copies per cell Natural way bacteria exchange genetic material within a species or between species Plasmids used as vectors 1. Made smaller (3Kb) 2. Selectable marker added (ampicillin resistance) 3. Polylinker or Multiple cloning site (many single RE sites) 4. Sometimes a scorable marker is added (Lac Z gene) white = inserted DNA blue = no insert Example: puc18 > Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 2

3 Restriction endonucleases (RE) Bacterial defense Phage (bacterial virus) Cut DNA at specific sites on the DNA endonuclease - cleaves DNA internally restriction - from its natural function restricts virus infection in bacteria bacteria Bacterial defense phage Cleaves at specific sequences of the phage DNA, but not the bact. chromosome because of methylation Example of RE HindIII - isolated from Haemophilus influenzae RE digests invading DNA bacteria Example of RE HindIII - isolated from Haemophilus influenzae bacteria third one identified strain Example of RE HindIII - isolated from Haemophilus influenzae bacteria third one identified strain recognition sequence (palindromic) 5 -AAGCTT-3 3 -TTCGAA-5 cleaves phosphodiester bond 3

4 Example of RE HindIII - isolated from Haemophilus influenzae third one identified strain recognition sequence (palindromic) bacteria 5 -A AGCTT-3 3 -TTCGA A-5 cleaves phosphodiester bond Example of RE HindIII - isolated from Haemophilus influenzae third one identified strain bacteria recognition sequence (palindromic) 5 -A AGCTT-3 ends are sticky 3 -TTCGA A-5 because of complementary base pairing cleaves phosphodiester bond Many types of REs 4, 5, 6, and 8 base pair recognition sequences smaller recognition sites are found more often in DNA, therefore produce smaller DNA fragments on average why? At any one base position, one of 4 bases 4 bp = 4 4 = 256 bp 6 bp = 4 6 = 4,096 bp DNA isn t completely random (patterns) Many types of REs can produce blunt ends, ex. HaeIII 5 overhangs, ex. HindIII 3 overhangs, ex. PstI DNA ligase T4 DNA ligase most commonly used Part of DNA replication Connects newly synthesized DNA fragments DNA repair Figure

5 Joining DNA fragments cut DNA from two sources with same RE denature just the ends and mix allow to anneal (i.e. complementary base pairing of sticky ends) results in recombinant molecules but not covalently linked DNA ligase is used to form new phosphodiester bonds between the fragments. Fig in text similar What do you do next? Bacterial transformation Have DNA ligated to a plasmid Next: transform E. coli Natural uptake of DNA from the surrounding environment Amplify cloned DNA by growing E. coli 13.7 The Polymerase Chain Reaction Makes DNA Copies without Host Cells 5

6 Section 13.7 Section 13.7 The polymerase chain reaction (PCR) copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities. PCR requires two oligonucleotide primers, one complementary to the 3' end of one strand of the DNA to be amplified and one complementary to the 3' end of the other strand. Section 13.7 The primers anneal to denatured DNA, and the complementary strands are synthesized by a heat-stable DNA polymerase (Figure 13.16). Figure Section 13.7 The three steps of PCR denaturation, primer annealing, and extension are repeated over and over using a thermocycler to amplify the DNA exponentially Recombinant Libraries Are Collections of Cloned Sequences 6

7 Section 13.8 Section 13.8 Ideally, a genomic library contains at least one copy of all the sequences in the genome of interest. Genomic libraries are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors. The choice of vector usually depends on the size of the genome. The number of clones in a library needed to give a certain probability of containing all genomic sequences is calculated as: N = ln(1 P)/ln(1 f) N is the number of required clones. P is the probability of recovering a sequence. f is the fraction of the genome in each clone. Section 13.8 Libraries can be made from subgenomic fractions such as a single chromosome generated by methods such as flow sorting (Figure 13.17) and pulsed-field gel electrophoresis. Figure Section 13.8 Section 13.8 A cdna library contains complementary DNA copies made from the mrnas present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mrna isolation. A cdna library is prepared by: isolating mrna from cells synthesizing the complementary DNA using reverse transcriptase cloning the cdna molecules into a vector (Figure 13.19) 7

8 Section 13.8 Reverse transcriptase PCR (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 Figure Section Specific Clones Can Be Recovered from a Library Probes complementary to part of a gene are used to screen a library to recover clones of a specific gene. Section 13.9 To screen a plasmid library, clones from the library are grown on agar plates to produce colonies. The 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 (Figure 13.20). Figure

9 Section 13.9 Cloning of diphenol oxidase The colony corresponding to the one the probe identified on the filter is identified and recovered. Colony Lift Success! Section 13.9 A phage library is screened by plaque hybridization. 9

10 Section Cloned Sequences Can Be Analyzed in Several Ways A restriction map establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment (Figure and Figure 13.22). Figure Figure ptacf3 (VspB-OxO) Section A Southern blot is used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest. 10

11 Section Southern blots can also be used to: determine whether a clone contains all or part of a gene ascertain the size and sequence organization of a gene or DNA sequence of interest (Figure and Figure 13.24) Figure northern blots look at RNA expression Canker margin tissue Figure Northern Blot A powerful tool to study gene expression DNA Sequencing Is the Ultimate Way to Characterize a Clone Used specific probe for diphenol oxidase 11

12 Section The most common method of DNA sequencing is dideoxy chain termination sequencing, developed by Sanger (Figure 13.25, Figure and Figure 13.27). Figure CGCTTTCA 5 Figure Figure Section Large-scale genome sequencing is automated and uses fluorescent dye-labeled dideoxynucleotides (Figure and Figure 13.29). Figure

13 Genetic engineering of eukaryotes Figure American chestnuts Choosing genes: Food for wildlife Example from restoration of the American chestnut tree safe, effective, & durable Agriculture & Forestry Chestnut Blight (Cryphonectria parasitica) Virulence factors of C. parasitica Cankering disease Mycelial fan formation Low ph at canker margin (ph2.8 vs. 5.5) : oxalate 13

14 Gene pyramiding Multiple disease resistance genes are more durable (less likely to loose effectiveness due to pathogen mutation) Why? If a gene has 1 in a million chance of being overcome, then 2 genes would have 1 in a million times a million (product rule) Durable (sustainable) With a single gene, resistance will usually be short lived unless the single gene has multiple functions Durable (sustainable) Two genes better than a single gene Durable (sustainable) Three genes better yet Extremely rare. Oxalate oxidase Gene comes from wheat Detoxifies oxalic acid: oxalic acid > H 2 O 2 + CO 2 Natural defense gene of grains Can enhance resistance to Septoria leaf spot in transgenic poplar ethylene glycol Other genes Chitinase Isolated from Trichoderma (a fungus that eats other fungi) Will degrade the chitin in C. parasitica s cell wall Therefore interfere with mycelial fan formation 14

15 p35s-cno self-processing pyramid vector Transforming American chestnut CaMV35S promoter (Signal peptide) Chitinase NIa OxO transcription/translation polyprotein export and processing (also reporter gene) Like bacterial transformation, only a small % of the plant cells get transformed Must be able to regenerate whole plants from a single, or a few transformed cells Tissues which can be regenerated are called totipotent To date, the only totipotent cells in chestnut are in immature embryos Transgenic chestnut How do we get the gene into the chestnut genome? Agrobacterium-mediated transformation Agrobacterium is a natural genetic engineer Agrobacterium infection in nature Inserts genes into plant genome which: Direct plant to produce opines (unique compounds only the specific Agrobacterium strain can metabolize) produce plant hormones that cause the formation of galls (or roots) in which the bacterium reside Interestingly - these inserted genes can only be expressed in plant cells, not the bacterium (how did they evolve?) Agrobacterium: bacteria that genetically engineers plants Vir genes 15

16 Agrobacterium-mediated transformation Use a binary vector T-DNA borders (allows movement into plant genome) surrounding useful genes Plant selectable marker (antibiotic resistance gene) Transgene with plant promoter and terminator Sometimes contains a reporter gene (ex. GUS) Outside T-DNA - bact. Selectable marker & origin of replication Agrobacterium mediated transformation Disarmed Agrobacterium strain Ti plasmid with T-DNA removed Ti plasmid retains vir genes which allow infection and transfer of T-DNA Agrobacterium mediated transformation Agrobacterium mediated transformation RB Selectable marker & transgene RB Only the DNA within the T-DNA Borders move into the plant genome LB LB Binary vector Disarmed Ti plasmid (containing vir genes) Binary vector Disarmed Ti plasmid (containing vir genes) Agrobacterium-mediated transformation How to make a tree from a seed, the hard way. Takes about 3 months Selection on medium with antibiotic ~2 week window (Dr. Xing based on S.A. Merkle) 16

17 Regeneration of whole plants Conformation of transformation Antibiotic resistance Or other selectable marker Polymerase Chain Reaction (PCR) DNA hybridization Reporter gene expression 24 month process! Biolistics - gene gun Transient gene expression Microscopic gold or tungsten particles DNA coated on the particles Particles shot into the cellular tissue DNA elutes off the particles and small % enters genome Gene gun Poplar leaf in oxalate oxidase assay Hemizygous transgenic plants Transgene in genome Any natural hemizygous genes? X-linked genes in males 17

18 Transgenic animals Transgenic animals Micro-injection (most common) Virus vectors Direct transformation Pharming Used to produce pharmaceutical products in animals (usually their milk) Example: clotting factor IX used to treat hemophilia Advantages: Prevent transmission of diseases (AIDS, hepatitis) Cheaper and more consistent supply Why milk? Few types of proteins, therefore easy to purify product Very strong and specific promoters available (example Casein gene promoter) Doesn t harm the animal because the product is localized in mammary glands variety of domestic mammals to use: cattle, sheep, goats, pigs, and mice Method Harvest egg and in vitro fertilize Microinject vector into male pronucleus Implant into foster mother Screen for transgenic offspring Only 1-2% of injected eggs produce transgenic offspring Gene Therapy In addition to gene products in milk, how about eggs? Germinal Can be inherited Totally transformed or mosaics As long as gonads are transformed Somatic Only affects individual Similar to an organ transplant Offspring do not carry the transgene 18

19 Example of Germinal Transgenic animals (germinal) Mice, sheep, goats, pigs, cattle, rabbits Chickens Fish Transgenic mice Gene encoding rat growth hormone Notice tails cut for PCR test Inject eggs Added a strong promoter to the salmon s growth hormone gene. All the same age 19.5 HUMAN GENE THERAPY Gene therapy is the introduction of cloned genes into living cells in an attempt to cure disease Research efforts in gene therapy is aimed to Alleviate inherited diseases Treat diseases such as cancer and heart disease Combat infectious diseases such as AIDS Refer to Figure 19.20a Nevertheless, some of the initial results are promising and future prospects abound Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display Carries a positive charge (cationic) Two transfer methods are used 1. Nonviral approach Human gene therapy is still at an early stage Figure Gene Therapy Involves the Introduction of Cloned Genes into Human Cells 2. Viral approach Refer to Figure 19.20b Virus is genetically altered so that it cannot proliferate after entry into host cells Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure Does not elicit immune response Low efficiency ADVANTAGE DISADVANTAGE High efficiency Elicits immune response

20 The First Human Gene Therapy Adenosine deaminase deficiency was the first inherited disease treated with gene therapy Adenosine deaminase (ADA) is an enzyme involved in purine metabolism If both copies of the gene are defective, deoxyadenosine will accumulate within the cells of the individual Deoxyadenosine is particularly toxic to B and T cells The destruction of these cells leads to a disease termed severe combined immunodeficiency disease (SCID) If left untreated, SCID is typically fatal at an early age Three approaches can be used to treat adenosine deaminase (ADA) deficiency 1. A bone marrow transplant from a compatible donor 2. Purified ADA coupled to polyethylene glycol (PEG) 3. Gene therapy On September 14, 1990 the first human gene therapy was approved for a girl with ADA deficiency Prior to the clinical trial, the normal ADA gene had been cloned into a retrovirus that can infect lymphocytes Figure outlines the protocol for the experimental treatment Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display This is called an ex vivo approach The genetic manipulations occur outside the body, yet the products are reintroduced into the body Discussion on GE Figure Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display After 10 months, immune responses were normal