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41 Cards in this Set
- Front
- Back
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Recombinant DNA |
Nucleotide sequence from two different sources (often diff species) combined in vitro (test tube) into the same DNA molecule |
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Genetic Engineering vs biotechnology |
Gen Eng: The direct manipulation of genes for practical purposes
Biotech: the manipulation of organisms or their genetic components to make useful products |
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DNA cloning |
methods for preparing that produce multiple copies of a gene or a DNA segment |
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Plasmids |
small circular DNA molecules that are replicated separately from a bacterial chromosome -only a small number of genes |
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Uses for cloned genes |
-making copies of (or amplify) a particular gene -producing a protein product |
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Cloning Vector |
DNA molecule that can carry foreign DNA into a host cell and replicate there (Plasmid) |
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Why are bacterial plasmids widely used as cloning vectors? |
1) readily obtained from commercial suppliers 2) easily manipulated to form recombinant plasmids 3) easily introduced into bacterial cells 4) multiple rapidly
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Steps in DNA cloning |
1. foreign gene of interest inserted into bacterial plasmid 2. plasmid becomes recombinant DNA 3. plasmid put back into bacterial cell - becomes recombinant bacterium 4. Recombinant bacterium is grown in culture 5. protein expressed 6. protein harvested 7. Research and Applications |
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List four applications of DNA cloning |
1. gene for pest resistance 2. bacteria for cleaning toxic waste 3. protein that dissolved blood clots in heart attack therapy 4. human growth hormone for stunted growth |
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Bacterial Restriction enzymes |
cut DNA molecules at specific DNA sequences called restriction sites, yielding restriction fragments |
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What is the most useful way for restriction enzymes to cut DNA? Why? |
staggered b/c it produces fragments with "sticky ends" stick ends bond with complementary sticky ends of other fragments |
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What is the role of DNA ligase? |
it is an enzyme that seals the bonds between the restricted fragments |
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Steps in using restriction enzyme to make recombinant DNA |
1. Restriction enzyme recognizes restriction site 2. restriction enzyme cuts sugar phosphate backbone 3. DNA fragment added from another molecule - cut by same enzyme 4. Base pairing occurs 5. DNA ligase seals strands |
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Gel Electrophoresis |
Indirect method of rapidly analyzing and comparing DNA fragments ** Unsure of this process- get someone to further explain |
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How does Gel Electrophoresis work? |
uses a gel as a molecular sieve -separates nucleic acids or proteins by size, electrical charge, or other properties -a current is applied and causes charged molecules to move through the gel -molecules are sorted into "bands" by size |
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PCR,purpose, and three steps |
Polymerase Chain Reaction Used for: production of DNA fragments, or method for genetic expression detection 1) Denaturation: -heating separates DNA 2) Annealing -Cooling allows primers to form hydrogen bonds with ends of target sequence 3) Extension/replication -heat resistant DNA polymerase adds nucleotides to the 3' end of each primer Cycle is continuous repeated. Numbers double with each cycle 2^n (where "n" is the number of cycles) |
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What is the key factor to successful polymerase chain reaction? |
A heat-stable DNA polymerase called Taq polymerase |
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PCR requirements? |
1) double stranded DNA (w target sequence) 2) heat resistant DNA polymerase 3) all four nucleotides 4) two 15-20 nucleotide primers (primers are the required starting point/catalyst for polymerase to bind on and attach base pairs) 5) Steps of denaturation, annealing, extension |
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DNA Sequencing |
1. DNA template strand 2. Add primer 3. labelled nucleotides are added - dideoxynucleotides. These do not allow for additional nucleotides to bind. nonlabelled nucleotides are added - deocynucleotides 4. denatured - separated from DNA template strands 5. Placed in capillary tube - for electrophorisis - shortest gets read first 6. laser reads labelled tags - sequence on spectrogram corresponds to DNA template sequence |
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what was the first automative method for DNA sequencing to be employed? |
dideoxy chain termination method |
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How does DNA sequencing differ from PCR? |
-only one primer is used -dideoxyribonucelotides are added -only copies one template -amplification is linear (not exponential) |
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Cloned genes can be expressed as proteins in either bacterial or eukaryotic cells, what are several difficulties that hinder the expression of cloned eukaryotic genes in bacterial host cells? |
1.Bacteria do not have DNA splicing machinery 2.Bacteria cannot perform post-transitional modifications which some proteins require |
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Bacteria do not have DNA splicing machinery - what key step must be taken to ensure successful cloning of eukaryotic genes in a bacterial host? How can this be accomplished? |
cloned in bacterial cells must only contain coding region (no introns) - includes only axons via cDNA creation |
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What is an alternative to bacterial host cells when cloning eukaryotic genes? |
Yeast -also may not possess the proteins required to modify expressed mammalian proteins but in this case mammalian proteins are used for study |
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Define Electroporation. What is it used for? |
-alternative to using vectors -applying a brief electrical pule to create temporary holes in plasma membranes -to introduce recombinant DNA into eukaryotic cells |
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What is an alternative method to vectors and electroporation? |
Inject recombinant DNA into cells using microscopically thin needles |
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Once recombinant DNA is inside a eukaryotic cell - how is the recombinant DNA incorporated into the cell's DNA? |
Through natural genetic recombination |
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Give an example of cross-species gene expression |
fly Pax-6 gene, given to a frog embryo, frog eye formed -two versions of the Pax 6 gene can substitute for each other -from an ancient common ancestor |
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What does the ability of the Pax 6 gene to substitute other versions of the gene tell us? |
a shared evolutionary ancestry of living species |
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Bacterial cells have differences in promoters and other DNA control sequences, how do scientists overcome this issue? |
Expression Vector -a cloning vector that contains highly active bacterial promoter just upstream of restriction site -the bacterial host recognizes the promoter and expresses the foreign gene that is now linked to promoter |
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Three methods for introducing recombinant DNA |
1. Vectors (Bacterial or eukaryotic cells - like yeast) 2. Electroporation (pulse to damage membrane) 3. injection using microscopic needle |
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Two types of DNA sequencing |
1. Dideoxyribonucleotide chain termination sequencing 2. Next generation sequencing |
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Describe how the two types of Next generation sequencing differ |
1)Sequencing by synthesis -single template strand is immobilized and amplified to enormous number of identical fragments -on nucleotide at a time. Chem technique can tell which of four nucleotides are added 2) Not fragments or amplified - Moving a single strand of DNA through a small pore in membrane - detecting bases one by one as they pass through and interrupt the current for a slightly diff length of time |
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First steps in understanding a cell type (ex embryo or cancer)? |
1. identify genes being expressed by cells of interest 2. identify mRNAs being made
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DNA technology used to study gene expression and function |
1. Nucleic acid probes - what cell is expressing 2. RT-PCR analysis - timing of expression 3. DNA microarrays - network of gene expression |
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Nucleic Acid Probes |
-probes can hybridize with mRNAs that are transcribed from gene -used to identify where a gene is transcribed in an organism 1) get identified gene sequence 2) synthesize probe with corresponding bases 3) In situ hybridization - using florescent dyes to tag probe for location of specific mRNAs in organism |
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Reverse Transcriptase-polymerase chain reacation |
-to compare gene expression between diff samples (ex: stages of development) 1)Reverse Transcriptase: create cDNA in vitro by using a single stranded mRNA as template with only EXONS 2) PCR - use cDNA template from reverse transcriptase. Add primers corresponding to segment of gene of interest. 3) Gel Electrophoresis: Products placed in gel to detect mRNA of interest |
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DNA microarray assays Define.. Improved technology of gene expression comparison by... |
Method of genome expression studies - network of gene expression -Compares patterns of gene expression in different tissues, at different times, or under different conditions 2)can measure the expression of thousands of genes at one time |
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Process of DNA microarray assays |
1. Isolate mRNA 2. make cDNA by Reverse Transcription 3. label nucleotides fluorescent 4. Apply cDNA mixture to microarray - diff gene in each spot 5. cDNA hybridizes with complementary DNA in microarray 6. wash of cDNA and scan for fluorescent -represents gene expressed
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What is one effective way of determining a gene function? |
Disabling gene and observe consequences ex: -in vitro mutagenesis -mutations are introduced to a cloned gene -mutated gene is reintroduced to cell -observe mutant's phenotype to develop normal gene function |
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How can you pinpoint the location of of a disease causing gene? |
Single Nucleotide polymorphism that is shared by ppl with disorder and not amongst unaffected people are genetic markers for a disease causing allele -occurs in every 100-300 base pairs -can be detected by PCR or microarray -SNP (marker) is always close to disease causing allele |
