exam 2 Flashcards
what is PCR and what are each of the 5 parts used for?
PCR (polymerase chain reaction): used to make many copies of a specific segment of DNA in a test tube (to amplify DNA segments)
- involves repeated cycles of heating and cooling and uses DNA polymerase to synthesize new DNA strands
ingredients required:
- DNA template: source of the DNA you want to copy (could be genomic DNA, a plasmid, or any other DNA source
- primers: short, single-stranded DNA sequences that bind to template DNA and specify the region of the DNA that needs to be amplified, act like bookmarks telling DNA polymerase where to start and stop copying
- DNA polymerase: the enzyme that actually makes the copies of the DNA, the one used in PCR is special because it can withstand high temperatures - taq polymerase (thermostable)
- dNTPs: building blocks of DNA, are added to the reaction mixture so that the DNA polymerase has raw materials it needs to make new DNA strands
- magnesium ions (Mg 2+): these ions help the DNA polymerase function properly
3 main steps of the PCR process
denaturation: reaction mixture is heated to separate the 2 strands of the DNA template (like unzipping a zipper)
annealing: temperature is lowered to allow the primers to bind to their complementary sequences on the template DNA (like matching up teeth of the zipper)
extension: temperature is raised again to allow the DNA polymerase to extend the primers and make new DNA strands (zipping up the zipper again)
these 3 steps are repeated many times (typically 20-40 cycles) and each cycle doubles the amount of target DNA
uses of PCR
used for genotyping: can be used to determine genotype of an individual by amplifying specific region of DNA and then analyzing the PCR product
- can be used to determine if a person has a specific mutation that causes sickle cell anemia
- to determine if a person is a carrier for a recessive genetic disorder, like cystic fibrosis
- can be used to amplify gene or DNA of interest which can then be inserted into a vector (like a plasmid) and cloned
- sequencing by using amplified DNA to determine the exact order of nucleotides
- crime analysis
- mutagenesis: introduce specific mutations into a DNA sequence
RT-PCR (reverse transcription PCR) + steps
used to measure the amount of RNA being produced from a particle gene + study individual splice forms
steps:
convert RNA to cDNA using reverse transcriptase and then PCR
analyzing sizes of PCR products would help determine which exons are included in the mRNA and therefore which splice form is being expressed
splice forms + how can they be identified
splice forms (also called is-forms: different version of mature mRNA molecules produced from the same gene due to alternative splicing
to determine splice forms: predicted from genome sequence, and measured by northern blots or RT-PCR (different splice forms appear as different sizes on the blot)
northern blotting
involves separating RNA molecules by size on gel, transferring to membrane, and hybridizing membrane with radioactive or fluorescent probes specific to gene of interest
used to determine: size, amount, and which RNA molecule is present
DdNTPs vs. dNTPS
DdNTPs lack a 3’ OH group and are used in Sanger sequencing
- when they are detected in Sanger sequencing, the DNA stops b/c no 3’ OH to bind to = various lengths to help determine which nucleotides are present
RFLP (restriction fragment length polymorphism)
technique that notices the variations in DNA sequences that create or destroy restriction enzyme recognition sites
- presence or absence of particular restriction site can be used to distinguish different alleles of a gene
- ex. sickle cell mutation destroys a restriction site for the enzyme DdeI = DNA from individuals with sickle cell anemia will be cut into different sized fragments by DdeI than DNA from individuals without the mutation
transposable elements
genes that hop around in the genome
- move by excising themselves from the genome, finding a new location, and splicing into it
transposase: enemy that recognizes host DNA & cut and glues transposons in and out of genes
Ac gene vs Dc gene
Ac gene: autonomous transposon
Dc gene: non-autonomous transposon
autonomous transposons encode their own transposes while non-autonomous require tranposase from somewhere else
if Ac is present in the gene, Dcs can be moved into the C allele = yellow kernels
- might also occasionally pop out Ds = purple spots
retrotransposons + 2 types
leave a copy behind when the jump out of the genome, causing mutations when they insert themselves into genes
- also make up 50% of the human genome
- thought to be evolutionary ancestors of retroviruses
2 types:
LINEs: autonomous transposons that can encode their own reverse transcriptase
SINEs: non- autonomous transposons that rely on enzymes from LINEs to move
RNA interference
natural cellular process that silences gene expression by targeting and degrading specific mRNA molecules
- triggered by presence of double-stranded RNA (dsRNA)
- uses small interfering RNA (siRNAs) and microRNAs (miRNAs form hairpin structures in the artificial approach to RNAi)
- RNAi can be passed down from generations in animals like C. elegans (defense mechanism against viruses)
quorum sensing
- bacteria secrete signaling molecules called autoinducers
- when population is high = autoinducer levels are high = triggers change in gene expression
- ex. biofilm formation in bacteria
one lone bacteria cant do anything but many together become an army
reporter genes
genes that are used to measure or detect gene expression in cells, tissues, or organisms
- attach reporter genes to genes of interest to monitor how much and when a gene is expressed
examples:
B-gal and GFP (green flourescent protein), luciferase (produces light in fireflies)
transgenes
foreign genes that are introduced and expressed in organism’s genome
examples: reporter genes, also transposons can act as transgender delivery systems
