Chapter 15 - Lecture/Book Notes- Genetic Analysis: an integrated approach PDF

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Lecture/Book Notes- Genetic Analysis: an integrated approach...

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Chapter 15: Recombinant DNA Technology and Applications (15.2, 15.3, 15.4) ⭐ Learning Objectives: Different types of recombinant DNA technology and their applications Transgenics, gene therapy, and cloning organisms mechanisms

15.1 Specific DNA Sequences are Identified and Manipulated Using Recombinant DNA Technology Recombinant DNA Technology: Recombinant DNA technology- for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo. ● Fragment and purify DNA into easily managed pieces ● Create copies of DNA fragments of identical sequence ● Combine DNA to construct recombinant DNA ● Determine the exact sequence of specific DNA ● Introduce specific DNA into organisms and assay the phenotypic effects

Restriction Enzymes (RE): Restriction Enzymes- Recognizes a specific DNA sequence and cuts both strands at that recognition site. ● Recognizes a particular sequence → cuts both strands of the sugar phosphate backbone of the DNA. ● First identified in bacteria → protects against viral infection ○ Restrict the growth of bacteriophages ○ RE identify/cleave viral DNA ○ Modification enzymes keep the host DNA methylated ● RE identify specific sequences and cut both strands of the sugar-phosphate backbone of DNA → producing either sticky or blunt ends

How does it protect its own DNA? Restriction-modification systems Restriction-modification systems- methylate the restriction site in bacteria, protecting bacterial sequences from digestion ● modify the restriction sequences in the bacterial DNA through the addition of methyl groups and thus protect the bacteria’s own DNA from being digested from endogenous RE.

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Sticky ends- single stranded segments with a 5’ or 3’ overhang produced by a staggered cut in the sequence. Termed “sticky” since they can stick to complementary base pair sequences via H-bonding. ● Produced by some RE cuts (ex. EcoRI) ○ Identifies palindromic sequences (same 5’-to-3’ base sequence) ● Principle: If two DNA molecules produced by restriction enzyme digestion have complementary sticky ends, they can be combined by complementary base pairing via H-bonding Blunt ends- Cuts that lack 5’ or 3’ overhangs/single stranded segments; still can be recombined but not through complementary bp. ● (ex. HindIII)

Restriction Maps: Data from restriction experiments to generate restriction maps of DNA sequences ● Restriction digests with one of more enzymes generate accurate maps. (Figure 15.1) Restriction mapping of lambda phage Purified lambda phage digestion: ● ApaI → 2 fragments/bands ApaI cuts genome once at one cut site and produce two fragments ● Xhol → 2 fragments/bands Xhol will cut at one cut site and produce two fragments ● ApaI+XhoI → 3 fragments/bands ApaI+XhoI both cut at their cut site once, producing three fragments RE cut at different sites produces accurate maps and genetic diversity.

Molecular Cloning: Isolated DNA fragments are inserted into a vector, to amplify the DNA ● Cloning vector- A piece of DNA derived from a plasmid, virus, or other biological source that can be stably maintained in an organism

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and into which heterologous pieces of DNA can be inserted. Many identical copies that result are called DNA clones. (Figure 15.3) Making recombinant DNA molecules

Plasmid cloning vectors replicate independently of bacterial chromosome and always remain separate ● Equipped with an origin of replication (ori) ○ Ori allows abundant DNA replication ● Contain genes for a trait that permits bacteria with the plasmid to be selectively grown ● Recombinant DNA amplified by repeated cycles of DNA replication

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(Figure 15.6) Plasmid cloning vector

(Figure 15.7) Amplification of recombinant DNA molecules in bacteria

Bacterial artificial chromosomes (BACs)- Cloning vector used in bacteria that utilizes the F plasmid origin of replication ● Have an insert size capacity of 100-200 kb (plasmids only up to 20kb) ● Used to generate DNA libraries DNA library- a collection of cloned fragments of DNA from a single source. ● Libraries derived from the genomic DNA of an organism are called genomic libraries ● Derived by mRNA are called complementary DNA (cDNA) libraries complementary DNA (cDNA)- Collection of DNA clones, originally derived via reverse transcription of mRNA molecules into DNA (cDNA) and cloned into a vector.

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Constructing DNA Libraries: (Figure 15.9) 1. Genomic DNA is isolated and fragmented into smaller pieces 2. Ligated into cloning vectors and transformed into bacteria 3. Collectively contain clones representing all sequences from the genome a. Different clones represent different parts of the genome b. When combined these clones form the whole genome mRNA sequences cannot be cloned directly for cDNA libraries

How similar do you expect genomic libraries and cDNA libraries to be? They are similar, and should be in both libraries since they are apart of the genome. How might this vary across tissues? cDNA captures expression and not all tissues are expressed at the same time. cDNA has more time-specific information. (Figure 15.10) Content of genomic vs cDNA Libraries

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The genomic library contains all the sequences cut at different sites The embryogenic cDNA library represents what is expressed during embryogenesis. The eye cDNA library are the genes that is only expressed in the eye.

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⭐15.2

Introducing Foreign Genes into Genomes Creates Transgenic Organisms Transgenic Organisms:

Transgene → another organism → transgenic organism Introduction of a gene from one organisms (transgene) into the genome of another organism creates a transgenic organism

Transgenic Bacteria:

(Figure 15.11) Typical Features of Expression Vectors

Expression vectors- vectors furnished with sequences capable of directing efficient transcription and translation of transgenes. - For transgenes to be properly expressed, the regulatory sequences must be compatible with the transcription and translation machinery present - Shine dalgarno sequence is important for translation in bacteria Codon bias- results from differences in which codons are used when there is more than one codon for a given amino acid. ● Interferes with expression due to relative scarcity of some tRNAs Production of Human Insulin in E coli ● Discovered by Fred Sanger in the early 1950s ● Recombinant bacteria produce insulin (through transgenic organism) ● Insulin hormone is a dimer of a A-chain and a B-chain which are linked together by a disulphide bond between two cysteine residues. ○ The A and B chains need to be combined to activate insulin Refer to (Figure 15.12) in the textbook 1. AA sequence of human insulin chain B was determined by polypeptide sequencing 2. Nucleotide sequence created by reverse transcription of AA sequence & addition of two

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successive stop codons to reading frame 3. Methionine codon inserted at the beginning of insulin B coding sequence to isolate insulin B protein 4. EcoRI & BamHI sites added to ends of DNA to facilitate cloning into vector 5. Insulin B chain cloned into cloning vector as a continuation of lacZ reading frame → creating fusion protein (expressed by lactose) 6. Protein produced by E.coli was purified → human insulin chain B separated from β-Gal by in vitro cyanogen bromide cleavage 7. Active insulin produced by mixing the two purified chains in an oxidizing atmosphere to induce disulfide bonds between cysteine residues of the two chains.

Transgenic Plants:

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Transgenic plants uses a natural plant transformation system from the soil bacterium Agrobacterium tumefaciens Ti plasmid- a large (200 kb) tumor-inducing plasmid ○ (Figure 15.13) ● Transfer DNA (T-DNA)- the part of the plasmid that is transferred from the bacterium to plant cell nuclei upon infection ○ Can undergo illegitimate recombination to integrate into the plant genome randomly. This causes plant cells to: (1) divide uncontrollably

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(2) produce amino acids only the bacteria can utilize as an energy source Genes in T-DNA can be replaced by a transgene of interest Totipotent = normal plant can be regenerated from a single isolated plant cell

Recombinant Technology to Produce Golden Rice: ● The edible part of rice lacks many micronutrients ● Endosperm synthesizes a precursor in carotenoids synthesis ● Five plant-derived enzymes are needed to produce β-carotene ● Single bacterial enzyme, CRTI, can replace three of these

(Figure 15.15) ● (b) The tumor genes are removed from the T-region to insert the bacterial CRTI gene that synthesizes B-carotene. ● (c)The phenotype of the transgenic plant is different than the wild type

Transgenic Animals:

Homologous recombination occurs less frequently than illegitimate recombination in animals ● Methods to introduce transgenes involve injection into eggs, embryos, or cells that give rise to gametes ● DNA can also be injected into cells that will subsequently be transplanted into an embryo and develop as genetic mosaics. Creating transgenic vertebrates involves injection of DNA directly into nucleus of a fertilized egg

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cell. ● Randomly integrates into genome by illegitimate recombination Production of transgenic vertebrates lead to variable transgene expression: 1. Integration of the transgenes as multicopy concatemers 2. Chromosome environment of the gene’s location Ex: Salmon: (Figure 15.17)

Ex: Mice (Mus musculus) Allows scientists to dissect many aspects of human development and physiology → as well as mouse development and physiology ● Targeted approach using homologous recombination (or CRISPR-Cas9-mediated genome editing) ● Non-targeted approach using illegitimate recombination Transformed mouse cells are cultured so that only those with the positive selectable marker and not the negative selectable marker will survive (positive-negative selection) (Figure 15.18) → Contains both knock-in and knock-out lines

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⭐15.3

Gene Therapy Uses Recombinant DNA Technology Gene Therapy:

Gene therapy- using genes to cure or alleviate disease symptoms Somatic gene therapy- targets cells whose descendants will not give rise to germ cells Germinal gene therapy- targets cells of the germ line, which give rise to gametes ● Therapeutic transgenes can be passed on to the progeny New somatic gene therapy makes it possible for gene editing in embryonic stem (ES) cells that corrects a disease-causing mutation. ● Fibroblasts can be reprogrammed in vitro to behave like stem cells → Induced pluripotent stem (iPS) cells ● Yamanaka factors act as pioneer factors to activate embryonic expression and reprogram epigenetic marks on the chromatin ● Use “disarmed” viruses to deliver/remove genes and gene products Manipulation in Vivo: (Figure 15.19) Bacteriophage Site-Specific Recombination Systems ● Ex: Cre-lox recombination system

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Example of Gene Therapy: Human Sickle Cell (Figure 15.20)

⭐15.4

Cloning of Plants and Animals Produces Genetically Identical Individuals Cloning of Plants and Animals: → producing genetically identical clones ● ●

Many plants can reproduce asexually → producing genetically identical clones from cells from a single organism Animals do not readily propagate clonally in nature (but there are some exceptions) ● Most animal cells are not totipotent, making cloning animals from single differentiated cells very complicated.

Dolly the sheep- the first cloned mammal (Figure 15.22) Cloning animals via Nuclear Implantation 1. Diploid nucleus isolated 2. Injected into an egg that had its nucleus removed.

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3. Created an embryo that was a genetically identical to the donor The only one of 270 injected eggs that resulted in the birth of a viable sheep. Case Study: Gene Drive Alleles (Figure 15.23) How genes can spread through populations

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