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Chapter 20 Biotechnology
Terms in this set (103)
methods of working with and manipulating DNA
DNA molecules formed whens segments of DNA from two different species are combined in vitro
the manipulation of organisms or their components to produce useful products
the direct manipulation of genes for practical purposes
methods for preparing well defined segments of DNA in multiple identical copies
what is a problem with DNA cloning?
naturally occurring DNA molecules are very long and have a lot of genes surrounded by noncoding sequences
small circular DNA molecule that replicates separately from the bacterial chromosome
the production of multiple copies of a gene
what are the two basic uses of gene cloning?
1. to make many copies of a particular gene
2. to produce a protein product
the typical gene makes up _____ of the DNA in a human cell
an endonuclease that recognizes and cuts DNA molecules foreign to a bacterium
specific nucleotide sequences that are characteristic of each restriction enzyme and is where they cut the DNA
What protects the DNA of a bacterial cell from its own restriction enzymes?
addition of methyl groups to adenines or cytosines within the sequences
most restriction sites are ____
the most commonly used restriction enzymes recognize sequences containing ______ nucleotides
4 to 8
a DNA segment that results form the cutting of DNA by a restriction enzyme
Steps of a restriction enzyme and DNA ligase making recombinant DNA
1. the restriction enzymes cleave the sugar phosphate backbones, forming sticky ends
2. DNA fragment from another source is added and the sticky ends of each base pair
3. DNA ligase seals the strands, forming a recombinant DNA molecule
a single stranded end of a double stranded restriction fragment
a linking enzyme essential for DNA replication, catalyzes the covalent bonding o the 3' end of one DNA fragment to the 5' end of another DNA fragment
what do two sticky ends form with each other to stay together (what type of bond)? which are....
hydrogen bonds, temporary unless sealed by DNA ligase
what has to occur for sticky ends to be formed?
the DNA from both sources has to be cut with the same restriction enzyme
a DNA molecule that can carry foreign DNA into a host cell and replicate there
what are the reasons why bacterial plasmids are used as cloning vectors? (4)
1. they can be obtained from commercial suppliers
2. they can be manipulated to form recombinant plasmids
3. they can be introduced into bacterial cells
4. they can multiple rapidly due to the high reproductive rate of bacteria
Steps in cloning the hummingbird gene using a bacterial plasmid (PART 1, 5 up to plasmid formation)
1. isolate hummingbird DNA and the plasmid with ampR and LacZ
2. cut both DNA samples with the same restriction enzyme (makes one cut in the plasmid lacZ gene and many cuts in the hummingbird DNA)
3. mix the cut plasmids and DNA fragments that allows base pairings between complementary ends
4.Add DNA ligase to seal them together
5. The DNA mixture is added to bacteria that have a mutation in their lacZ gene
Steps in cloning the hummingbird gene using a bacterial plasmid PART II
1. plating out the bacteria on solid nutrient medium with ampicillin allows us to distinguish the cells that have taken up plasmids
2. the presence of x-gal in the medium allows us to distinguish the colonies with recombinant plasmids from those with non recombinant plasmids (lacz breaks down x-gal and turns blue)
3. left with white colonies that represent hummingbird DNA
the cloning procedure which starts with a mixture of fragments from the entire genome of an organism and no single gene is targeted
a set of cell clones containing all the DNA segments from the original genome, each within a plasmid, or other cloning vector
How are bacteriophages used as cloning vectors?
fragments of foreign DNA are spliced into a trimmed down version of a phage genome and the normal infection process allows production of new phage particles each carrying the foreign DNA insert
Bacterial artificial chromosome (BAC)
a large plasmid that acts as a bacterial chromosome and can carry inserts of 100,000 to 300,000 base pairs
that standard plasmid carries ________ base pairs
what is an advantage and disadvantage of using a BAC?
1. the large insert size minimizes the number of clones needed to make up the genomic library
2. the size makes them harder to work with in the lab
a double stranded DNA molecule made in vitro using mRNA as a template and the enzymes reverse transcriptase and DNA polymerase
steps in making complementary DNA from eukaryotic genes
1. reverse transcriptase is added to a test tube with mRNA
2. Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT's as a primer
3. mRNA is degraded by another enzyme
4. DNA polymerase synthesizes the second strand
5. results in cDNA which carries complete coding sequences
What allows reverse transcriptase to begin creating DNA off of the RNA primer?
since mRNA has a poly A tail, the reverse transcriptase can use a strand of thymine deoxyribonucleotides (tDs) as a primer
a gene library containing clones that carry complementary DNA inserts
what is the advantage of a genomic library?
1. if you want to clone a gene but don't know what cell type expresses it or cannot obtain cells of the right type
2. If you are interested in the regulatory sequences or introns associated with a gene, a genomic library is necessary
advantages of a cDNA library
1. can study a specific protein in a cDNA library
2. can be used to study sets of genes expressed in particular cell types
nucleic acid hybridization (think probe)
the process of base pairing between a gene and a complementary sequence on another nucleic acid molecule
nucleic acid probe
in DNA technology, a single stranded nucleic acid molecule used to locate a specific nucleotide sequence in a nucleic acid sample
how are probes usually labeled?
radioactive or fluorescent
a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted
what are two problems with expressing cloned eukaryotic genes in bacteria?
1. the presence of introns make the gene long and unwieldy so they cannot be expressed correctly
2. the bacterial cells do not have the cell machinery to modify the genes after translation
what is the advantage of using yeasts instead of bacteria hosts for cloning?
1. they are as easy to grow as bacteria
2. they have plasmids
a brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membranes through which DNA can enter
polymerase chain reaction
a technique for amplifying DNA in vitro by incubating it with specific primers, a heat resistant DNA polymerase, and nucleotides
steps of the polymerase chain reaction
1. during each of the three cycles, the reactant mixture is heated to denature and separate the DNA strands
2. the mixture is cooled to allow annealing of single stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence
3. a heat stable DNA polymerase extends the primers in the 5 to 3 direction
what is the heat resistance DNA polymerase called?
what is a benefit of PCR?
only a minute amount of DNA needs to be present in the starting material, it can even be partially degraded
how do you calculate the number of target segment molecules created in PCR?
2^n, n is number of cycles
why can't PCR amplification substitute for gene cloning?
1. Occasional errors during PCR replication impose limits on the number of good poise that can be made
2. PCR imposes limits on the length of DNA fragments that can be copies
a technique for separating nucleic acids or proteins on the basis of their size and electrical charge affecting their rate of movement through an electric field in a gel made of agarose
how do nucleic acids get separated in the gel in gel electrophoresis? (2)
1. they carry negative charges on their phosphate groups so they all travel towards the positive pole
2. as they move, the agarose fibers impedes long molecules more than shorter ones (by length)
restriction fragment analysis relies on ______
the band pattern characteristic of the starting molecule and the restriction enzyme used
variations in DNA sequence among a population
restriction fragment length polymorphism
a single nucleotide polymorphism that exists in the restriction site for a particular enzyme thus making the site unrecognizable by that enzyme and changing the length of the restriction fragments formed
what is a disease that can utilize restriction fragment analysis and why?
sickle cell disease, it is caused by a mutation of a single nucleotide within a restriction sequence so the analysis can distinguish normal and sickle cell alleles based on band pattern
a technique that enables specific nucleotide sequences to be detected in samples of DNA involving gel electrophoresis of DNA molecules and their transfer to a membrane followed by nucleic acid hybridization with a labeled probe
steps of southern blotting in differentiating a heterozygous sickle cell from a homozygous normal (5)
1. preparation of restriction fragments (each mixed with the same restriction enzyme)
2. gel electrophoresis forms different patterns of bands
3. DNa transfer (blotting) produces a blot with a pattern of DNA bands like the gel
4. hybridization with labeled probe that is complementary to the gene of interest (beta glob in gene)
5. Probe detection with photographic film that detects the radioactivity
what is gene sequencing carried out by? based on what?
sequencing machines based on the dideoxyribonucleotide chain termination method
steps of the dideoxy chain termination method (colored tags)
1. the fragment of DNA to be sequenced is denatured into single strands and incubated with the ingredients for DNA synthesis
2. synthesis of each new strand continues until a dideoxyribonucleotide is inserted at random instead of the normal deoxyribonucleotide which prevents further elongation of the strand
3. eventually, a set of labeled stands of various lengths is generated with a colored tag representing the last nucleotide of the sequence
4.the labeled strands are separated by passing through a gel where the shorter strands move through quicker
in situ hybridization
a technique using nucleic acid hybridization with a labeled probe to detect location of a specific mRNA in an intact organism
what is the basic strategy in genome wide expression studies?
1. isolate mRNAs made in particle cells
2. use mRNAs as templates for making corresponding cDNAs
3. employ nucleic acid hybridization to compare this cDNA with a collection of DNA representing all or part of the genome
4. results identify the subset of genes that are being expressed at a given time
DNA microarray assays
a method to detect and measure the expression of thousands of genes at one time
how do DNA microarray assays work?
1. tiny amounts of a lot of single stranded DNA fragments are fixed to a glass slide in an array
2. isolate mRNA and make cDNA from it
3. apply cDNA mixture to the microarray where it will hybridize with any complementary DNA
4. rinse off excess cDNA and loo for fluorescence
in vitro mutagenesis
a technique used to discover the function of a gene by cloning it, introducing specific changes into the cloned genes sequence, reinserting the mutated gene, and studying the phenotype of the mutant
a technique used to silence the expression of selected genes that uses synthetic double stranded RNA molecules that match the sequence of specific genes to trigger the breakdown of the gene's mRNA or block its translation
genome wide association studies
a large scale analysis of the genomes of many people having a certain phenotype or disease with the aim of finding genetic markers that correlated with the phenotype or disease
DNA sequences that vary in the population
what is the most useful genetic markers?
single base pair variations (single nucleotide polymorphisms)
single nucleotide polymorphisms
a single base pair site in a genome where a nucleotide variation is found in at least 1% of the population
True or false: SNPs usually contribute to the diseases their hosts have
False; most are in noncoding regions but are located close to a disease causing allele, and since little crossing over takes place between regions so close, scientists can use it to locate the gene
the division of an asexually reproducing cell into a collection of genetically identical cells
cloning that produces organisms genetically identical to a parent who donated an initial cell
describing a cell that can give rise to all parts of the embryo and adult
remove the nucleus of an unfertilized or fertilized egg and replace it with the nucleus of a differentiated cell; if the nucleus from the differentiated donor cell retains its full genetic capability then it should be able to direct development of the recipient cell into all the tissues and organs needed
why did scientists have to resort to nuclear transplantation in cloning?
because differentiated cell from animals do not divide in culture so they used nuclear transplantation to determine whether differentiated animal cells were totipotent
in cloning ____ is inversely related to _____
the potential of a transplanted nucleus to direct normal development (success of nucleus), age of the donor
what concluded that the nucleus does change as animal cells differentiate?
Briggs & King's frog experiment; the older the donor nucleus they transplanted, the lower percentage of normally developing tadpoles (nuclear potential becomes more restricted as embryo develops)
cloning with the goal of producing new individuals
true or false: cloned animals always look or behave identically
False (EX human twins are always slightly different)
why do only a small percentage of cloned embryos develop normally?
1. in the nuclei of fully differentiated cells, a small subset of genes is turned on and the rest are repressed by methyl groups
2. these changes have to be reversed from a donor animal for transfer and need chromatin restructuring which occurs incompletely during cloning procedures
3. misplaced methyl groups in the DNA of donor nuclei may interfere with gene expression necessary for normal embryonic development
any relatively unspecialized cell that can produce, during a single cell division, one identical daughter cell and one more specialized daughter cell that can undergo further differentiation
many early animal embryos contain stem cells in the ______ stage
blastula / blastocyst
adults stem cells
stem cells that serve to replace non reproducing specialized cells as needed (like nerve cells)
what is a difference between embryonic and adults stem cells?
adults stem cells are not able to give rise to all cell types in the organism though they can generate multiple types
what is the ultimate aim in scientific medical stem cell use?
repairing damaged or diseased organs
describing a cell that can give use to many but not all parts of an organism
Embryonic stem cells are
when the main aim of cloning is to produce ES cells to treat disease
How are Induced Pluripotent Stem cells made?
researchers transformed differentiated cells into ES cells by using retroviruses to introduce extra cloned copies of four stem cell master regulatory genes so they act like stem cells
what are two major potential uses for human iPS cells
1. cells from patients suffering from a disease can be reprogrammed to become iPS cells and act as model cells for studying the disease
2. a patient's own cells could be reprogrammed into iPS cells and used to replace nonfunctional tissues
How can scientists use PCR to track down pathogens and diseases?
they can diagnose disorders by using PCR with primers that target the genes associated with these disorders
the introduction of genes into an afflicted individual for therapeutic purposes
why does gene therapy have the potential for treating disorders traceable to a single defective gene?
a normal allele of the defective gene could be inserted into the somatic cells of the tissue affected by the disorder
steps of gene therapy using a retroviral vector (4)
1. insert RNA version of normal allele into retrovirus
2. let retrovirus infect bone marrow cells that have been cultured from the patient
3. viral DNA carrying the normal allele inserts into chromosome
4. inject engineered cels into patient
what has to happen for gene therapy of somatic cells to be permanent? give example
the cells that receive the normal allele must be ones that multiply during the patients life (bone marrow cells)
explain "Synthesis of Small Molecules for Use as Drugs"
finding the sequence of proteins needed by tumors to survive led to the identification of molecules that combat cancers by blocking the function of those proteins
what is an example of a Small Molecule for Use as Drugs
imatinib inhibits receptor tyrosine kinase that is needed in leukemia
what were the first two protein (that are in the body in small amounts) pharmaceutical products manufactured
human insulin and human growth hormone
pertaining to an organism whose genome contains a gene introduced from another organism of the same or a different species
Concept of "Pharm" animals
scientists use whole animals to produce large quantities of proteins by introducing a gene from an animal of one genotype into the genome of another animal of a different species
steps of making a transgenic Pharm animal
1. scientists remove eggs from a female of the recipient species and fertilize them in vitro
2. cloned the desired gene from the donor
3. inject the cloned DNA directly into the nuclei of the fertilized eggs
4. the engineered embryos are implanted into a surrogate mother
4. the gene encodes a protein desired in large quantities
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