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89 Cards in this Set
- Front
- Back
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Mutation |
A change in DNA sequence that can be inherited - they are gene dependent - they are random and rare (2-12 x10^-6 per gene per gamete) |
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Mutant |
An organism that experiences a change in DNA sequence |
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Wild Type vs. Mutant |
Wild Type - considered to be the norm - more frequent - first Mutant - displays a negative effect |
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Phenotype change |
Any type of change we can track Altered appearance, growth conditions, behaviour, molecules, etc. |
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Types of Mutations |
Small Changes based on nucleotides: - Base-pair substitutions - Insertions - Deletions - Inversions - Translocation Large changes, chromosome rearrangements: - Insertion - Deletions - Inversions - Translocation - Reciprocal Translocation - Duplication - Genome duplication |
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Base-pair substitutions (2 types) |
Transition: Purine -> Purine or Pyrimidine -> Pyrimidine 2 types Transversion: Purine <--> Pyrimidine 4 types of changes |
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Inversions |
Rotate strand 180 degrees - so the 5' and 3' ends match |
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Fluctuation test |
To see whether mutations are responding to a specific stimulus or just random
- Add selective agent penicillin to some cell cultures, only mutants can survive If mutations occur in response to stimulus, the same number of cells will survive in each tube If mutations occur over time and randomly, some test tubes will have a lot of mutants and some will have none Test results show that mutations are random |
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Replica plating test |
To detect all the cells of a certain phenotype - put colonies of penicillin sensitive bacteria onto one master plate - velvet imprint to "photocopy' the pattern and the cells - put replica plates (with different media) on velvet to pick up the cells in the same distribution - wait to see if they replicate, whichever colonies do are penicillin resistant - we know that the mutation was present in the colonies for sure now |
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How do spontaneous mutations occur? |
Depurination Deamination * Breaking of DNA backbone (X ray) UV light (pyrimidine dimers) Mistakes during replication* Unequal Crossing OVer Slippage (unstable trinucleotide repeats)* |
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Deamination |
- Amino group in cytosine lost, becomes carbonyl and thus uracil CG -> UG After two rounds of replication, 2 WT (CG), 1 mismatch (UA) and one mutant (AT) Causes a base pair substitution: GC to AT transition - must be in germ line cell to be passed on to the next generation |
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Mistakes during Replication |
Causes a base pair substitution (either transition or transversion) - can be fixed by 3' to 5' exonuclease ability of a DNA polymerase |
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Slippage |
- happens in runs or repeats during replication - after one round of replication one will be WT and the other will be insertion/deletion If newly synthesized strand slips -> insertion after another round of replication If template strand slips -> deletion after another round of replication Huntington's Disease (of nervous system) - due to slippage in trinucleotide sequence of gene Huntintin - jerky movements, mental decline, behavioural Fragile X Syndrome - due to slippage in trinucleotide sequence of gene FMR1 on X chromosome - autism, intellectual disability |
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Mutagen |
Alters DNA |
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Induced Mutations |
Replace a base: base analogs Alter a base structure or property: - add an OH - add a methyl - remove an amino group Insert between bases: intercalating agents Radiation: X-ray, UV Biological agents: transposons, virus |
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Endogenous vs. Exogenous |
Endogenous: mutations caused by the inside - nucleotide imbalances - metabolic processes going wrong - repair mechanisms going wrong Exogenous: mutations caused by the outside - any chemical that changes DNA (but toxicity is not equal to mutagenicity) |
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Sunlight: spontaneous or induced mutation? |
Spontaneous: walking outside normally Induced: above and beyond what you experience normally, i.e. you forget your sunscreen |
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The Hulk: spontaneous or induced mutation? |
Induced - he was irradiated by gamma rays! - mutation probably insertion/deletion - mutation depends on environment (i.e. when he gets angry) |
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Repair systems for mutation |
- proofreading - base excision repair - nucleotide excision repair - methyl-directed mismatch repair Basic rule of thumb: 1. initial recognition by enzyme specific to type of damage 2. damage directly repair or removed (enzymes) 3. if removed, causes a small/large abasic site 4. Gap repaired by DNA polymerase 5. DNA ligase connects backbone of DNA |
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How to classify mutations |
Based on origin: Spontaneous vs. induced Based on molecular change: Base substution, insertion, deletion, etc. Based on effect on translation: silent, missense, nonsense, frameshift Based on effect on function |
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Silent mutation |
Change in base at wobble position (3rd) so amino acid isn't changed - amino acids encoded by 1-6 codons |
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Missense mutation |
Mutation that exchanges one amino acid for another (First or second position) Severity of effect depends on what substitution and where |
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Nonsense mutation |
Mutation that causes a stop codon UAA, UAG, UGA - results in a truncated protein Severity of effect depends on where the mutation occurs |
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Frameshift mutation |
Frameshift mutation - insertion or deletion of 1 or 2 nucleotides - results in wrong protein sequences Severity of effect depends on where the mutation occurs |
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Mutations in non-coding regions |
Increased/decreased protein levels No protein Misexpressed proteins (wrong time or wrong space) |
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How do mutations affect function? |
Allele dependent, NOT gene dependent! - different alleles carrying mutations in the same gene can be loss/gain of function alleles Often hard to predict effect of a mutation on function, experimentally analyze the mutants Loss of function alleles: - null or amorphic - hypomorphic - incomplete dominance - dominant loss of function - dominant negative loss of function Gain of function alleles: - hypermorphic - neomorphic |
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Recessive vs. Dominant loss of function mutations |
Recessive: Heterozygote is WT, Recessive homozygote is mutant Dominant: Heterozygote is mutant, Recessive homozygote is mutant |
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Loss of function mutation - null allele and amorphic allele |
Null - no protein synthesis, stop codon early on Amorphic - type of null, protein is so deformed it can't function Heterozygotes are WT, Homozygotes for mutation are mutant ex. Arabidopsus, single bp sub -> no gametes or reproductive organs |
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Hypomorphic allele - loss of function mutation |
Reduced protein synthesis, or protein is somewhat deformed and thus has weak function - often mutation in promoter, or in the gene Heterozygotes are WT ex. Eyeless in Drosophila, pax6 in mouse, aniridia in humans |
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Incomplete dominance - loss of function mutation |
intermediate levels of gene products cause intermediate phenotypes |
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Dominant loss of function mutation and haplo-insufficiency |
Haplo-insufficiency - one WT allele is not sufficient for a WT phenotype (property of the WT allele) So mutants thus cause loss of function - reveals dosage-sensitive genes ex. T locus, tail length in mice |
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Dominant negative loss of function mutations (antimorphic) |
These mutations antagonize WT function - interfere with or block the WT function ex. one allele codes for 1/2 a dimer, one half of dimer mutated and can't bind to regulatory sequences, no transcription of another gene |
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Gain of function mutation: hypermorphic alleles |
Increased protein synthesis or a protein that functions better (ex. enzyme with higher activity) - often dominant - Heterozygotes mutants ex. possum allele codes for sodium channel, lets more sodium through, mouse can't turn over if laid on the back |
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Gain of function mutation: neomorphic allele |
Very rare! - mutation that causes protein to gain a new function - or ectopic expression: express a protein where its not usually expressed ex. Homeotic mutations: one type of organ replaced by another (Antennapedia in Drosophila) |
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Restriction Enzymes - what they are - types |
Specific enzyme cuts specific recognition sequence - can differ in length - result in specific cutting patterns Blunt ends Sticky 5' ends (5' overhang) Sticky 3' ends (3' overhang) Fragments only fit back together (+ ligation) if they have: - same type of ends - matching sequences |
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Ligation of: - blunt ends - sticky 5' and 3' ends |
Blunt ends fit with any other blunt ends - if you mix and match you lose the restriction site Different 5' sticky ends can be ligated if they have the same overhang, lose restriction site |
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Length of restriction sequences and length of digest (complete vs. incomplete) |
4 bp will cut every 4^4 = 256 bp 6 bp will cut every 4^6 = 4096 bp 8 bp will cut every 4^8 = 65.5 kb Choose length of recognition sequence and length of digest to control: - average fragment length - number of fragments |
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Gel electrophoresis and its effects on Plasmid vs. Genomic DNA |
- DNA fragments are separated by size - DNA negatively charged, on agarose gel - small fragments migrate faster Plasmid DNA - few fragments, distinct bands Genomic DNA - many fragments, smear |
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Restriction Mapping |
Divide solution with cloned DNA into 3 portions Digest each portion: 1 with EcoRI, 1 with BamHI, and 1 with both Load each digested sample into gel, along with size markers in another lane Can analyze the gel electrophoresis results to make a restriction map with the cut sites of the two restriction enzymes |
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What a vector must contain to be used successfully for cloning |
Origin of Replication (ex. ori in E.coli) Selectable marker (ex. ampicillin) Restriction site (at least one) |
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Vector vs. Plasmid |
Vector: self-replicating DNA molecule that can be used to transfer DNA between host cells, and whose presence can be detected Plasmid: extra chromosomal DNA originally found in bacterial species |
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Steps to create a genomic DNA library |
- Cut DNA of interest and vectors with the same restriction enzyme (creates compatible ends) - Generates a population of vectors, each one with a different part of the genomic DNA - transform vectors into E.coli (via electroporation, must be free of all salts?) - select for transformed cells via ampicillin |
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Polymerase Chain Reaction - what you need - steps |
Need: - target DNA - template DNA - DNA you start with (plasmid, genome, restriction fragment, PCR fragment etc.) - two different primers that are complementary to the 3' end of each strand of target DNA, 18-25 nucleotides Steps: 1. 94C for 5 mins - Purify and denature DNA from target source - add to solution with primers, Taq DNA polymerase and deoxynucletide triphosphates 2. 50-60 C for 30 seconds - Primers base pair at sites flanking target sequence 3. 72C for 1-5 mins - Polymerization from primers along templates
- thermocycling - expontential copying (after 33 rounds -> millions of copies) |
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PCR - what can you amplify? |
Anything! - coding/regulatory - complete/partial - known/unknown - if you include a mismatch in the primer you can change the sequence |
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Reverse Transcription |
RNA template, reverse transcriptase synthesizes DNA - results in a hybrid molecule (mRNA strand and cDNA strand) |
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Steps to make a cDNA library |
1. Remove all mRNA in an organism and purify 2. Add add polydT primer, treat with reverse transcriptase and dATP, dCTP, dGTP, and dTTP 3. Denature cDNA-mRNA hybrids and digest mRNA with mRNAase. 3' end of cDNA folds back on itself and acts as a primer 4. First cDNA strand acts as a template for synthesis of second cDNA strand with dNTPs and DNA polymerase (5. PCR?) 6. Insert generated fragments into plasmid |
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Genomic libraries vs. cDNA libraries |
Genomic DNA fragmented by restriction digest or shearing - fragments almost equally represent the complete sequence cDNAs are represented according to expression pattern and expression levels |
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Hybridization - different types - general method |
Types: Southern Blot - DNA as a template - digest and size separate fragments Northern Blot - RNA as a template - size separate General method: - soak gel in alkali solution to denature DNA - blot into nitrocellulose membrane - place membrane in bag or tube - add labelled (radioactive, DIG) DNA probe (denatured) - probe binds to complementary sequences but does not have to be perfect match - see where probe binds |
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Primer vs. Probe |
Primer: - small - is extended - usually not labelled - amplification - you buy them Probe: - large - does not extend - needs to be labelled - no amplification - you make them BOTH anneal to complementary sequences |
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Sanger sequencing |
Denature template DNA Use ONE labelled primer, add ddNTPs and polymerase - different ddNTPs in each reaction mix - 4 reactions - load each onto a gel and then separate fragments by size Automated version - fluorescent labelled ddNTPs, results in chromatogram |
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Positional Cloning |
Finding a gene sequence based on its phenotype - can be done with and without mapping information Hemophilia A - no mapping info (requires family history and an idea of what is causing the mutation) Cystic fibrosis - mapping info needed |
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Hemophilia A |
- coinherited with colour blindness -> genes are close on the chromosome - family history helped determine it was X-linked, recessive, single gene - Mutation that inactivated Clotting factor VIII (involved in cascade that forms blood clot) |
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Normal blood clotting cascade if you don't have Hemophilia A |
Cleaving of coagulation factor II into thrombin (active form) - Thrombin is a serine protease that converts fibrinogen (soluble) into fibrin (insoluble) - fibrin is a fiber that gets crosslinked by factor VIII to form a blood clot |
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Process of Hemophilia A cloning |
Purify Factor VIII Obtain amino acid sequence Reverse translation into coding sequence (computer, because of wobble position?) Synthesize degenerate (similar but not identical) oligonucleotides - pool of probes Create probe library Also use human genomic DNA library Colony Hybridization (Probe probably won't latch onto whole gene sequence, especially if sequencing shows few nucleotides) Shearing: break genomic DNA, get fragments that overlap each other to get sequence of whole gene- Make probe with the new sequencing info Repeat colony hybridization |
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Colony Hybridization |
- Take vectors from genomic library and plate them - Velvet imprint so that you have a reference plate - take original plate and do a nitrocellulose membrane lift, hybridize with radioactive probe prepared from degenerate probe sequences, see the probe's location - find that same location on the reference plate and grow those cells to isolate the plasmid with a DNA fragment |
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Hemophilia A gene characteristics found |
On X chromosome 186 kb long 26 exons Complicated! |
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To prove that they found the right gene for Hemophilia A... |
- genes from Hemophilia A patients were sequenced and compared to the wild types - identified mutations: base substitutions, splice mutations, small and large deletions |
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Cystic Fibrosis |
Affects mostly European children Autosomal recessive gene Many symptoms, die early (before 30) Thick mucus in bronchioles (obstructed airway), more susceptible to bacterial infection |
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Process of cystic fibrosis cloning |
Used mapping info to build a linkage map to a known sequence Chromosome walking and Chromosome jumping to get to CF gene |
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Mapping to a close known sequence (linkage mapping) |
Collection of markers of known map position on the chromosome that cover portion of genome Use second set of markers for better analysis (these markers could be a known sequence, restriction site, restriction polymorphism) Try to find markers as close as possible to the predicted gene |
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Chromosome walking |
Restriction digest a fragment
Prepare a radioactive probe Use the probe in a colony hybridization with a human genomic DNA library Identify a colony containing a vector with matching sequences Sequence the human genomic DNA fragment in the vector Use above info to make the next probe Reprobe the library to find another human DNA fragment with the matching sequence Go down the chromosome this way |
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Chromosome jumping (simplified) |
Restriction digest human genomic DNA Ligate the fragments into circles - known sequence next to unknown sequence at the other end Use a sequencing primer for the known sequence, it'll start sequencing into the unknown area Now you can skip everything in between (the jump!) and go onto the next fragment! |
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Chromosome walking vs. Chromosome jumping And how they work together |
Jumping - allows you to go longer distances - can get around a sequence that is difficult to sequence or clone Walking - continuous - no sequences missed You can combine the two methods... - then you might find some ORFS = candidate genes - compare in wildtypes and mutants to see if differences in the gene |
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CF gene characteristics - what it codes for and how it works |
Codes for ABC protein - chloride ion channel - important for the secretion of sweat and mucus |
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- problem with relying or morphological mutants that mutated spontaneously - advantage to induced mutations |
Problem - too few of them - they happen too slowly - we need mutants in more ways than morphological (subtle changes like biochemical pathways) Advantage to induced: - increased frequency - larger variety |
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Ways to induce mutations |
Ethyl methanesulfonate (EMS) - popular mutagen Transposon mutagenesis Targeted Knockouts |
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Ethyl methanesulfonate (EMS) |
Popular mutagen Alkylating agent Transfers its ethyl group to Guanine, disrupting 1 of its 3 H-bonds -> can only form 2 H-bonds -> Guanine starts mispairing with Thymine Causes GC to AT transition Mutagen is easy to use - soluble, can be taken up by cells Expect 1 mutant in 1000 individuals You need to screen individuals for any type of mutant (morphological, developmental, behaviour, biochemical, reactions to environment etc.) Using plants is easy because you can save the seeds - animals you need to take care of and keep alive |
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Problems with EMS |
Can't predict the location of the mutations No selection Often more than one mutation per mutant Single base pair change somewhere in the genome - hard to track on a molecular level |
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Complementation Assay |
Use EMS and identify mutants with the same phenotype - need to figure out if they carry a mutation in one gene or more than one Start with homozygous mutants (most mutations recessive so mutants often homozygous) - one for each mutant - mutant makes non functional gene If the mutation is in the same gene, crossing them will cause all F1 phenotypes to be mutant (No Complementation) If the mutation is in two different genes (assuming the genes are unlinked) all F1 phenotypes will be wildtype heterozygotes (mutations can complement each other) |
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Transposon Mutagenesis (no detection) |
"Jumping genes" - gene (known DNA sequence) in a plasmid that inserts randomly into the genomic DNA - often modified so they only jump once - gene also contains selectable marker (ampicillin or kanamycin) - wildtype plants are killed - 1 living mutant out of 1000 dead individuals - don't have to search for mutants like with EMS - but transposon will be in different locations in the genome -> generate a lot of different mutant lines from each plant - don't screen individuals for mutants - instead you select among the mutants for helpful mutations - multiple transposons can jump into one genome |
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Transposon Mutagenesis vs. EMS mutagenesis |
EMS - look for 1/1000 mutants among individuals - screen individuals to find the mutants - use phenotypic analysis Transposon - look for 1/1000 live mutants among dead wildtypes - select among mutants for beneficial mutations - cannot just use phenotypic analysis - can also use a "molecular tool box" because we inserted a known fragment of DNA |
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How to detect the transposons in the genome - purpose - process |
Purpose: distinguish if we have one copy or two - is the phenotype due to one of the copies or both? (we already know the transposon sequence) Process - isolate genomic DNA - digest with RE (probably will cut on the left and right of the transposon) - size separate on a gel - Southern Blot with a transposon specific probe (designed with transposon sequence) |
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Transposon sequence primer design |
- complementary to transposon (known sequence) - also design so primers sequence outward into genome (unknown sequences) - helps identify the sequence that has been mutated by the transposon - allows you to correlate molecular basis to the phenotype much more quickly |
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Transposon mutagenesis: - if both gene and transposon sequence are known - finding transposons in specific genes |
Use gene specific primer and a transposon specific primer - gene primer sequence towards transposon, transposon primer sequence out into gene - PCR using isolated genomic DNA - should only get a band in the gel if these two primers are in close proximity -> transposon in gene of interest |
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Problems with Transposon Mutagenesis |
- insertions are random - can't target an insertion to a specific gene Could try Targeted Mutations with recombination constructs, but homologous recombination is not as effective in some species |
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Targeted Knockouts |
Destroy a specific gene - can only be done in specific organisms, and need to know the sequence of the gene - NOT random like EMS and transposon mutagenesis (they don't require the sequence of the gene) - works well in yeast, hard in mouse, not in Drosophila, Arabidopsis - works in zinc fingers, CRISPR, TALENS - "tailoring genes" - introduce bp changes Process - clone the known gene sequence (construct), put the selectable marker in the middle of the gene (knockout constructs) - selectable marker selects for the sequence and destroys the original gene function - homologous recombination events happen (i.e. crossing over) between construct in plasmid and genome - results in an exchange of DNA between construct and genomic DNA - wild type sequence in plasmid, selectable marker ends up in specific gene - test the selectable marker to ensure the switch was made |
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Polymorphic locus |
A locus with two or more alleles where each allele is present in more than 1% of the populations - they are called "genetic variations" not WT or mutant Examples: SNPs (single nucleotide polymorphisms) Indels SSRs (simple sequence repeats) CNPs (copy number polymorphisms) Complex variants (none of the above) |
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Locus |
Any location within the genome with a defined chromosomal location - irrespective of function - coding or noncoding - short or long - single bp substitution, one gene, several genes |
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Allele |
Genetic variation at a specific locus |
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SNPS (single nucleotide polymorphisms) |
Most common genetic variation Can be spontaneous (during replication) or induced (chemical mutagen) - base pair substitutions |
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How to detect SNPs |
1. Southern blot analysis of restriction site-altering SNPs - probe for the intervening sequence between two restriction sites - one R site lost in allele 2, on the autoradiogram homozygous allele 2 will have one larger piece of DNA (homozygous allele 1 smaller piece, heterozygote two bands) 2. PCR analysis of restriction site-altering SNPs - two primers surrounding the restriction site (one complementary to 3' top strand, and one complementary to 3' bottom strand) - PCR both alleles and gel electrophoresis - homozygous 1 has 2 small pieces, homozygous 2 has one big piece, heterozygous has 3 bands 3. Allele-specific oligonucleotide hybridization - SNP probe to a known SNP sequence (short so that it'll only bind to the one allele) |
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Sickle Cell Anemia |
Single bp substitution - single amino acid change in Beta globin chain of hemoglobin - promotes the aggregation of hemoglobin under low oxygen, distorting RBCs to sickle shape - heterozygotes resistant to malaria |
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Effect of Temperature on Primers |
Increase in temp to denature DNA, and then decrease temp to allow primers to anneal (50-60C) If primers are perfect match, they'll anneal at higher temperatures. If primers are close but not perfect match, they'll only anneal at lower temperatures. If you use the same genomic DNA and the same primers, but vary the temperature - more mispriming will occur at lower temperatures (more bands in electrophoresis) - allows for identification of gene families (use probe to find similar sequences in another species) |
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Southern probe vs. SNP probe vs. Primer |
Probe not primer - not extended - no amplification - labelled Primer and SNP probe - you buy them - small All three anneal |
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SSRs aka Microsatellite DNA |
Result of faulty replication - polymerase slips because the sequences are identical, organisms end up with different numbers of repeats (highly polymorphic in length) - associated with disease (triplet repeats - Huntington's, fragile X, Myotonic dystrophy) - During replication, strands occasionally reanneal out of register, one strand is longer than the other - DNA repair of template strand so now both strands are longer |
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Detection of SSRs |
Use PCR to detect repeat length polymorphisms - primers must be located outside of repeat sequence - one will be shorter and the other will be longer - heterozygotes will have the lengths of each allele on gel electrophoresis |
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Huntington's and SSRs |
CAG triplet repeat region (CAG = glutamine) - more repeats = disease and the earlier it comes |
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Minisatellite DNA |
subcategory of SSRs - repeats of more than 3 nucleotides - can be large (500 bp to 20 kb) - at a smaller number of genomic loci - differ between individuals - used for "fingerprinting" - very low chance that two unrelated individuals will have the same genotype |
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Process of fingerprinting with minisatellite DNA |
Isolate genomic DNA from different individuals RE digest - sites outside of repeat sequences Size separate on a gel Southern Blot (probe for the repeat sequences) Autoradiogram is unique for each person, compare to DNA from crime scene |
