BIOTECHNOLOGY Campbell and Reece Chapter 20 DNA Technology

BIOTECHNOLOGY Campbell and Reece Chapter 20

DNA Technology �made possible: • Human Genome Project completed in 9 yrs (2001) • by 2010 genomes of > 7, 000 species

Recombinant DNA �DNA formed when segments of DNA from 2 different species are combined in vitro (in test tube) �useful for analyzing genes & gene expression

Biotechnology �manipulation of organisms or their components to produce useful products �includes selective breeding, using bacteria in fermentation

Genetic Engineering �the direct manipulation of genes for practical purposes

DNA Cloning �useful for researcher to have just that portion of DNA working with • 1 gene may be as small as 1/100, 000 th of a chromosome �bacterial plasmids: circular DNA molecules that replicate separately from bacterial chromosome • used by bacterium when environment changes

Gene Cloning

Restriction Enzymes �used to cut DNA w/in short, specific nucleotide sequences (restriction sites) set of dbl stranded DNA fragments with single stranded “sticky ends”

DNA Ligase �sticky ends form H-bonds with sticky ends of C’ bases from other DNA: temporary bonds �DNA ligase make bonds permanent: makes covalent bonds in sugar-phosphate backbone

Restriction Enzymes Animations � http: //www. dnalc. org/resources/animations/rest riction. html � http: //highered. mcgraw- hill. com/olcweb/cgi/pluginpop. cgi? it=swf: : 535: : /sites/dl/free/0072437316/120078/bio 37. swf : : Restriction%20 Endonucleases � http: //www. dnalc. org/view/15476 -Mechanism- of-Recombination-3 D-animation-withbasic-narration. html

Cloning Vectors �name given original plasmid �dfn: a DNA molecule that can carry foreign DNA into host cell & replicate there �bacterial plasmids mostly used because 1. readily available from suppliers 2. can insert foreign DNA in vitro bacterial cell 3. multiply rapidly

Cloning Vectors Animation �http: //highered. mcgraw- hill. com/sites/0072556781/student_view 0/cha pter 14/animation_quiz_2. html

Storing Cloned Genes in DNA Libraries

Bacterial Artificial Chromosome (BAC) �large plasmids trimmed down so they contain just the genes necessary for replication �carry 100 – 300 kb (kilo base pairs), normal plasmid can insert vectors no larger than 10 kb

c. DNA can make “libraries” using c. DNA:

Genomic & c. DNA Libraries �each have advantages �Genomic library good if: • looking for a gene but not sure where it is in a genome, or what kind of cell to look in • if looking for introns or regulatory sequences as’c w/gene �c. DNA library good if studying: • specific protein • sets of genes expressed in particular cell types • changes in patterns of genes over life of cell (during development of organism)

Screening Library for Clones Carrying Gene of Interest �Nucleic Acid hybridization: process of base pairing between a gene & a C’ sequence on another nucleic acid molecule �C’ molecule = ss. DNA or ss. RNA = nucleic acid probe �probe is made that is C’ to known sequence in gene

Nucleic Acid Hybridization

Detecting a Specific DNA Sequence by Hybridization with a Nucleic Acid Probe

Expressing Cloned Eukaryotic Genes �several technical difficulties hinder the expression of cloned eukaryotic genes in bacterial hosts �can substitute eukaryotic hosts: yeasts, some insect cell, some mammalian cells that have appropriate expression vectors: a cloning vector that contains a highly active bacterial promoter just upstream of restriction site where eukaryotic gene can be inserted allowing gene to be expressed in bacterial cell or have been genetically engineered to use in specific eukaryotic cells

PCR �Polymerase Chain Reaction �amplifies specific target segment of DNA in vitro using primers that bracket the derived sequence, & a heat-resistant DNA polymerase, & nucleotides

PCR

�http: //www. dnalc. org/view/15475 -The- cycles-of-the-polymerase-chain-reaction. PCR-3 D-animation-with-no-audio. html

What DNA Technology Allows �once you have many copies of a gene you can ask questions about its functions, where & when the gene is expressed, or how important is it to the organism

Gel Electrophoresis �uses a gel made of a polymer (agarose commonly) �gel acts like molecular sieve �separates nucleic acids or proteins based on charge and size �nucleic acids carry (-) charges on phosphate group so (+) end of gel �as move the longer molecules impeded more by “sieve”

Gel Electrophoresis

Restriction Fragment Analysis �fragments produced be restriction enzymes put thru gel electrophoresis band pattern characteristic of starting molecule & restriction enzyme used �able to identify viruses & plasmids by their band patterns �then recover DNA from gels � 1 way of getting pure samples of DNA – won’t work with DNA from eukaryotic cells: gel electrophoresis smear not bands

RFLP �Restriction Fragment Length Polymorphism: single nucleotide polymorphism (SNP) that exists in the restriction site for a particular enzyme, making the site unrecognizable by that enzyme & changing lengths of the restriction fragments formed by digestion with that enzyme �found in coding & noncoding DNA

Using restriction fragment analysis to distinguish the normal & sickle cell alleles of the human β-globin gene

Southern Blotting �used to detect certain nucleotide sequences w/in a complex DNA sample �compares the restriction fragments produced from different samples of genome DNA

Southern Blotting

Dideoxy Chain Termination �method used to sequence relatively short DNA fragments �done by automated sequencing machines

Dideoxy Chain Termination �technique: synthesizes a set of DNA strands C’ to original DNA fragment • each strand starts with same primer & ends with dideoxyribonucleotide (dd. NTP) • incorporation of dd. NTP terminates growing DNA strand because it lacks 3’ –OH group (site of attachment of next nucleotide) • each dd. NTP tagged with distinct fluorescent label so identity of nucleotide at end of each strand (ultimately entire strand) is identified

Northern Blotting �in vitro hybridization with labeled probes looking for specific m. RNAs �could be used to look at how expression of a gene changes during the embryonic development of organism �carry out gel electrophoresis on m. RNA

RT-PCR �Reverse Transcriptase- Polymerase Chain Reaction • quicker & more sensitive than Northern blotting • isolates m. RNA from different developmental stages of organism then add reverse transcriptase to make c. DNA which serves as template for PCR amplification using primers from gene being studied • bands will be in samples that originally contained the gene being studied

in situ hybridization �alternative method used to determine which cells are expressing certain genes �done in living organism �probes labeled with fluorescent dyes

DNA Microarray Assays

DNA Microarray Analysis �uncover gene interactions �suggest correct therapeutic route in cancer treatments

in vitro Mutagenesis �most common method used to determine function of gene: disable it & observe what happens �specific mutations introduced to cloned gene & then mutated gene returned to cell knocking out normal gene in the process

RNAi �RNA interference �method for silencing expression of selected genes �uses synthetic ds. RNA molecules matching the sequence of gene to trigger breakdown of the gene’s m. RNA or to block its translation

RNAi �important when studying groups of genes to determine how multiple genes interact (basis of systems biology, chap 21) �in humans considered unethical to block activity of genes

Genome-Wide Association Studies �used to analyze genomes of large #s of humans with certain phenotype or disease �test for genetic markers: DNA sequences that vary in a population • uses SNPs (single nucleotide polymorphisms) • single base pair site where variation is found in at least 1% of population • few million in human genome

Using SNPs

�as Cloning advances being made in DNA technology also working on technology to make multicellular organism from 1 cell producing genetically identical organism � 1 st attempted late 1950’s

Cloning �Genomic Equivalence: an organism’s cells have the same genome • proved when able to generate new organism from 1 cell • used carrot (root) cells cultured adult plant

Single-Cell Cultures �totipotent: describing a cell that can give rise to all parts of the embryo & adult, as well as extraembryonic membranes in species that have them

Nuclear Transplantation �used in cloning animals �transplant nucleus from a differentiated animal cell enucleated ova can sometimes give rise to clone

Cloning Animals

Stem Cells �Embryonic Stem Cells (ES) or Adult Stem Cells from animal embryos or adult tissues can reproduce & differentiate in vitro and in situ �ES cells are pluripotent: cell that can give rise to many but not all parts of an organism • difficult to acquire

Stem Cells �ES cells currently donated by patients undergoing infertility treatment or from long term cell cultures established with cells isolated from donated embryos �when main objective is to produce ES to treat disease process called therapeutic cloning

Stem Cells �now scientists can “de-differentiate” cells returning them to pluripotent cells: called i. PS: induces Pluripotent stem cells • can do anything ES cells can do

i. PS Cells � 2 1. • • major uses reprogram cells from patients with disease to become i. PS cells then act as model cells for studying the disease & potential treatments Parkinson’s disease, type 1 diabetes field of regenerative medicine 2. • patient‘s own cells used to regenerate damaged tissues

Gene Therapy �introducing genes into afflicted individual (into somatic cells) for therapeutic purposes �useful for disorders caused by single gene defect (overall, relatively small # of all diseases) �for it to be permanent, treated cells must be the ones that continue to divide thru out patient’s life

Gene Therapy

Forensic Uses of DNA Technology �using Short Tandem Repeats (STRs) in DNA isolated from crime scenes leads to genetic profile • strong evidence to prove suspect innocent or guilty • used in paternity disputes • identification of remains

Transgenic Organisms �gene for desired protein inserted into bacterial genome and become tiny “factory” for making protein • Insulin • Digestive enzymes • Growth Hormone

Environmental Clean Up �genetically engineered microorganisms developed for oil spills or to degrade toxic waste materials • Bacteria • Algae • Plants

Transgenic Plants & Animals �to improve productivity & food quality