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Gene technology
animal health technology-vet techs
| Question | Answer |
|---|---|
| What are exons in genes? | -coding part |
| What are introns in genes? | -non coding part |
| Does mRNA show exons or introns or both? | -only shows exons |
| how can DNA mutations arise? | -spontaneously -or by DNA damaging units |
| What are the 5 DNA damaging units? | -promoter -5' UTR - non synonymous changes - synonymous changes -3' UTR -intergenic changes |
| What do promoters do? | -change in transcription |
| 5' UTR? | -alter translation efficiency |
| Non-synonymous? | -alter amino acids and maybe protein function |
| synonymous? | -do not alter amino acids |
| 3' UTR? | -alter mRNA stability or transport |
| Intergenic change? | -no observable effect |
| Splice site mutation? | -insertion into an intron before an exon causes mRNA to incorrectly process it and splice out that exon |
| DNA to protein process? | -DNA to transcription using rRNA to mRNA to translation using tRNA to protein |
| Sampling collection? | -extract DNA = PCR -extract RNA = reverse transcriptase PCR for DNA sequencing, SNP genotyping, microsatellite genotyping, agarose or capillary electrophoresis or restriction endonuclease + agarose electrophoresis |
| Sampling for genetic tests? | -hair -blood -semen -faeces -buccal swabs -tissues post mortem in ethanol |
| Where is DNA found? | -in nucleated cells -RBC, WBC, hair roots (but not hair shafts), tissue |
| How is DNA stored? | DNA is stable -tissue stored in ethanol -blood stored in EDTA tubes and chilled -dry hair stored in plastic bag at room temp |
| How is RNA stored? | RNA is unstable and needs specialist storage -stored by freezing immediately at -80 degrees Celsius in liquid nitrogen or storage solutions |
| Ways of contaminating a sample? | -contamination by bacteria or viruses -cross contamination of samples -contamination by collector's DNA -contamination by collector's skin enzymes which degrade the sample remember PCR can amplify very small contaminants |
| How to prevent contamination of samples? | -sterile environment: clean gloves, swabs etc -change gloves between samples -label samples |
| What is the purpose of PCR? | -to make millions of copies of the gene that you want to analyze |
| Steps of PCR? | -Denaturation: 1minute at 94 degrees C to separate two strands -annealing: 45seconds 54 degrees C, 2 short oligos added and bind to target region strands -extension: 2minutes at 72 degrees C, enzyme Taq DNA polymerase adds dNTP's to oligos, now 2DNA |
| How many times is the PCR cycle repeated? | -30 to 40 cycles for one gene-test |
| agarose gel electrophoresis? | -samples added to wells and an electric current is applied that moves the fragments to the positive terminal -bigger fragments will situated towards the top as they move slower -smaller fragments will be closer to the positive terminal as faster |
| how is agarose gel read? | -a dye is applied that binds to the nucleic acids and fluorescents under UV light either SYBR or ethidium bromide |
| Cant test mRNA, why? | -as it is constantly being broken down |
| Real time PCR compared to transcript PCR? | -improves speed and see things as it happens in real time -is very sensitive for very small particles and specific -don't have to run a gel |
| What is the CT value? | -threshold cycle, when dye is strong enough to read due to enough replication produced and begins exponential phase |
| SYBR green advantages? | advantages -intensity is proportional to amt of PCR products produced -can monitor the amplification of ds-DNA |
| SYBR green disadvantages? | -non specific, primers can bind to something else and it comes up green, generating false positive results -can use melt curve analysis after PCR to confirm if target was amplified by melting curve which is dependent on sequence |
| Real time PCR probes? | -bind to specific target sequences -bind in between PCR primers -have two ends a dye (fluorophore-short) and quencher (fluorophore-long) that are released when polymerase extends primers; and they produce a fluorescence |
| Control gene? | -expressed in all cells -used as a comparison -gives the relative difference in the amount of DNA or RNA |
| What are microsatellites? | -simple sequences of tandem repeats -found throughout the genome -highly polymorphic they have many alleles (many alternative forms of a gene) due to high mutation rates |
| How do microsatellites mutate? | -by replication slippage -the new strand becomes a different length to the template -normally arises in their closest sizing counterparts |
| what is the microsatellite mutation rate? | 10^-3 to 10^-4 per locus/per generation compared to normal eukaryotic DNA sequence 10^-9/nucleotide per generation |
| What does the probability that two individuals will have the same genotype at multiple loci randomly; depend on? | -no. of alleles -freq of alleles -no. of loci |
| why do we want to identify individuals? | -forensics (human, wildlife and domestics) -trade in wildlife and wildlife products -non invasive monitoring of populations |
| Parentage testing for microsatelites? | -extract DNA by hair or blood -amplify loci by PCR -separate alleles by electrophoresis to get multiple loci (fingerprint) -parentage by exclusion |
| Parentage by exclusion? | -can the mother be the mother, ie. can she contribute one allele at each locus to the offspring -can the father be the father -does the mating qualify |
| Accurate parentage information allows what? | -identification of carriers of recessive alleles -avoids inbreeding -management strategy -parentage information on a large farm -verification of semen or embryos purchased -guaranteed pedigree purchase of studs |
| What are SNP'S? | -single nucleotide polymorphism -more efficient as can measure on large scale and cost effective -still rudimental for animals but potential for parentage testing -it is a mutation in the middle of nowhere when common and doesn't do anything |
| SNP's compared to microsatellites? | -lower mutation rate due to testing larger numbers of loci -less homoplasy-identity by state not descent |
Created by:
sherloki
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