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28 Cards in this Set
- Front
- Back
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The Songer Chain- Termination Method of DNA Sequencing
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1. Primer is bound to template strand
2. Reaction ingredients added 3. Primer extension, Chain termination when a ddATP is inserted 4. Newly synthesized strands recovered, loaded on gel in the A lane. The primer is shown as a black block. |
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Restriction Mapping of a cloned Fragment of DNA
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1. Population of cloned DNA fragments is prepared
2. DNA fragments are cut w/restriction enzymes 3. The restriction fragments are separated by gel electrophoresis 4. Theoretical models are constructed that are consistent w/results 5. Models are tested against results of double enzyme digests 6. Conclusion: model 1 is correct |
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Example of a sequencing gel
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Fig 17-18
The fragments at the bottom of the gel are the shortest (sequences closest to primer) Each fragment reading from the bottom of the gel to the top is one nucleotide longer than the fragment below it The gel is read from bottom to top to determine the sequence of the DNA |
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Use of Fluorescent Labels in Automated Sequencing
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1. Primer added
2. Reaction ingredients added 3. Primer extension chain termination product recovery. |
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Polymerase Chain Reaction (PCR)
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These 3 steps (denaturation, annealing, extension) are performed for 25-30 cycles and can give greater than 1x10^6 fold increase in the amt of DNA
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Advantages of PCR
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1. Fast- Thermocyclers can perform 25 cycles in about 2 hours
2. Extremely Sensitive DNA from a single cell can be used as the template 3. Oligo primers are cheap and can be synthesized quickly |
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Limitations of PCR
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Must know the sequence of the target DNA in order to make the oligo primers
Because of the high level of sensitivity, it is easy to get contaminating DNA in the sample. |
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Characterizing Cloned DNA (1)
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The nucleic acid sequence of the gene can be derived:
Compare deduced protein sequence to actual protein sequence Compare sequence of the gene to sequence of known genes to try to derive a fxn for that gene |
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Characterizing Cloned DNA (2)
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The clone can then be used to study the sequences of the regulatory region of the gene:
This is possible only for genomic clones because cDNA clones just contain coding sequences |
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Characterizing cloned DNA (3 &4)
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3. The clone can be used to isolate similar genes from other organisms: Use as a heterologous probe
4. If the gene is of clinical importance, the clone can be used for diagnostic or therapeutic purposes: e.g. Mutations in breast cancer genes (BRCA1/BRCA2) |
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Characterizing Cloned DNA: Restriction Mapping
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Restriction mapping: one of the first steps in characterizing a newly cloned piece of DNA
DNA clone is cut w/different restriction enzymes;determine sizes of each fragment by comparing w/known "size standards" by agarose gel electrophoresis Map deduced from all single and double digest products |
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Restriction Fragment Length Polymorphism (RFLP)
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When different alleles of a gene differ in restriction enzyme digestion patterns
Very useful in studying genetic diseases |
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Cloning in Eukaryotic Cells
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Yeas Saccharomyces cerevisiae- Eukaryotic cell, but grown like bacteria, so it is easy to grow and manipulate
Useful for protein expression of cloned genes: most eukaryotic proteins must be post-translationally modified Safe organism to use for producing proteins used for therapeutics and vaccines. |
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Using Yeast to Express Human Recombinant Proteins
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Yeast have been used to express large amount of human recombinant proteins for use in therapeutics and vaccines
Table 17.1: Recombinant Prot: Hepatitis B virus surface protein Malaria parasite protein Insulin |
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Polymerase Chain Reaction (PCR)- part a
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PCR is a cell-free system to amplify large amts of a specific region of traget DNA
Short oligonucleotide primers are used to replicate a particular target DNA sequence |
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3 Basic Steps involved in PCR
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1. Denaturation: the target DNA strands are "pulled" apart by heating the samples at ~95 C to produce single stranded DNA
2 .Annealing: the temperature is lowered to bw 50-70 C to allow the primers to anneal to the template 3. Extension: a heat-stable DNA polymerase (usually Taq) copies the template in a 5' to 3' direction using the annealed primers as the starting point to produce double-stranded DNA |
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A simple cloning vector
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pUC18:
ampicillin resistance gene lacZgene: polylinker region |
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Cloning Using a Plasmid Vector
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puC18 plasmid w/intact lacZ gene can metabolize Xgal and will form blue colony
Polylinker cut w/restriction enzyme Recombinant plasmid cannot metabolize Xgal and will form white colony |
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Cloning DNA using Plasmid Vectors
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Plasmid vector is removed from bacterial cell and cut w/a restriction enzyme & The DNA to be cloned is cut w/the same restriction enzyme
The two DNAs are ligated to form a recombinant molecule Introduction into host cell. Cells carrying recombinant plasmids can be selected or screened by plating on medium containing antibiotics or color indicators such as Xgal |
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Cloning Vectors
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Carriers of cloned DNA:
plasmids bacteriophage DNA bacterial artificial chromosomes (BACs) yeast artificial chromosomes (YACs) mammalian viruses (gene terapy vectors) |
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Different ends produced by different restriction enzymes
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Ex:
Smal --> blunt ends Pstl --> 3' overhang sticky ends BamHI --> 5' overhang sticky ends |
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Ligation of DNA Digested with EcoRI
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Cleavage w/EcoRI:
Fragments w/complementary tails Annealing allows recombinant DNA molecules to form by complementary base pairing. The two strands are not covalently bonded as indicated by shaded gaps. DNA ligase seals the gaps, covalently bonding the two strands. |
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Restriction Enzymes cut DNA at specific sites
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e.g. Hind III from Haemophilus infuenzae strain R:
Palindrome: nucleotide-pair sequences that read the same forward or backward from a central axis of symmetry Ex Restriction Enzyme: EcoRI |
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What is cloning and why do we clone genes?
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once a gene has been cloned, its fxn can be investigated using many new molecular techniques:
1. nucleotide sequence can be determined 2. Deduce amino acid (protein) sequence from the nucleotide sequence Determined protein fxn: fxnl studies and comparison to other proteins 4. Study expression pattern of the gene: where and when is it expressed? how is expression regulated? 5. If the protein has potential therapeutic applications, it can be expressed at high levels and purified. |
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HOw do we clone and study genes?
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1. Purify DNA from an organisms (or from cells grown in culture)
2. Restriction Enzyme Mapping: Enzymes that recognize a particular DNA sequence and cut the DNA at that sequence 3. The restriction fragments are ligated into carrier molecules (vectors) to make recombinant DNA (rDNA) 4. The rDNA molecules are transferred to a host cell (e.g. E. coli) by transformation: vectors replicate, producing large amounts of the rDNA molecule that can then be purified for further studies 5. The cloned DNA can be expressed to produce protein for various applications. |
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What is cloning?
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A gene must be cloned in order to manipulate it using Recombinant DNA technology.
Cloning at the organismal level: producing an exact genetic duplicate of the organism: asexual reproduction in eukaryotes produces clones |
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Molecular cloning
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replication of DNA fragments (usually specific genes) with the aid of plasmid or viral "vectors" in an appropriate host cell:
produces large quantities of cloned genes, use to characterize the gene and the gene produce (protein) encoded by that gene Also can produce large amts of the protein that can be used for therapeutic purposes. |
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What is Recombinant DNA technology?
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Technique used to produce new combos of DNA in the lab that are not normal found in nature
Allows for: isolation, replication, and characterization of genes and gene products, easy manipulation/modification/expression of genes of interest (Genetic Engineering): producing new variations of an organism, express genes not normal expressed in that organism e.g. pesticide and disease resistant crops Recombinant DNA techonolgy has many important application in our society: production of medicines, diagnosis of diseases, genetic engineering of plants and animals, and criminal investigations |
