Flashcards in Gene Analysis Deck (19):
Uses a DNA or RNA probe to detect DNA fragments separated by gel electrophoresis
Genome structure, mutations, copy number, DNA fingerprinting, RFLP linkage analysis (LS4), evolution
Uses a DNA probe to detect RNAs separated by gel electrophoresis
Tissue specific expression, gene regulation
DNA digest of genomic DNA
if i want to know if there is a similar gene between organisms, we do genome extraction, isolate dna and run it on a gel. You get smears becuase dna broken up into big and small pieces (bands not from pcr). Then you stain with EtBr and do a southern blot
Southern blot process: get the DNA onto nitrocellulose paper
Unlabeled DNA cut with restriction enzyme and run on a gel, fragments separated by electrophoresis
Transfer dna onto a filter paper. (Alkaline solution, sponge, agarose gel, nitrocellulose filter paper, paper towels, with weight on top).
Alkaline solution moves up through gel and sponge which lifts dna off gel and onto nitrocullolose paper, paper towels on top wick solution all the way through
Southern blot process: finding a specific sequence
Take foodsaver bag, take off nitrocellulose paper and put in the bag with a buffer and a probe. Could be radioactivly labeled with base added with a kinase or flouescent, then incubate it.
Rinse of extra fluid and expose it onto film (radioactivity) or visualize fluorescence, labeled DNA probe hybridized to complimentary DNA bands
Southern blotting and comparing genomic structure
Southern blotting can be used to compare genomic structure to cDNA
If you wanna know about the region where the dna lies, if using cDNA probe, you know regions of cDNA are complimentary to the exons
cut genomic DNA with restriction enzyme and you get fragments of different sizes
do the southern blot you get 3 bands and this tells you that there are three exons in the gene (best guess one band per exon)
How can southern blots reveal mutations?
If there is a mutation it can be in one allele but not the other. There are different length fragments it one allele versus another in a single individual. Can see what alleles have a mutation/deletion in genes by looking where the bands are compared to the wild type
How can a southern blot predict gene copy number?
Neu oncogene is amplified in some breast cancers
Correlates with poor prognosis
Run genomic DNA on gel, blot and probe for neu
Tumors with amplified neu show a more intense band
How does northern analysis identify in which cell type a gene is expressed?
Identify specific mRNAs by hybridization to a DNA probe:
Isolate mrnas (polyt)
separate on aragose gel
transfer to nitrocellulose
use DNA probe to see in which tissue the mRNA is expressed
How can northern blots measure the expressed RNA from a gene?
Isolate mRNA at different times after the induction of differentiation
Separate RNA on a gel
Probe with gene
Ex: beta globin gene expression is induced when red blood cells differentiate from precursor cells. Take sample at different times to see when genes are turned on or turned off. Can see how much maturation needed before gene is turned on
Can take all of white colonies(our library), sample them, put in tube with primers(use primers that are in the plasmid sequence).
Genomic library of plasmid has introns and exons, on either side of insertion site there is a known sequence, where the primers are.
Take each colony and take a little with pipette and put it in growth fluid, grow it so you get enough copies, take sample and do an extraction, isolate plasma dna using known fragments (and primers generate from known fragments) to generate more copies via pcr. Then you can sequence it- Have to sequence a lot of inserts to get the whole genome.
Based on chemical degradation of DNA
Sanger or Dideoxy Sequencing
Based on premature termination of DNA synthesis using dideoxynucleotides which cause termination of strand elongation- no 3' OH no nucleophilic attack of next base
Process of Sanger /Dideoxy Sequencing
DNA to be sequenced is denatured
Oligonucleotide of known sequence is hybridized to template DNA
Template primer is distributed in 4 tubes
Different dNTPS and ddNTPs are added to each tube (elongating and terminating nucleotides). Need correct ratio
In vitro DNA synthesis produces collection of fragments, each terminating with the dideoxynucleoside
How do you visualize the bands in Sanger/dideoxy sequencing?
Fragments denatured and separated on polyacrylamide gel and visualized by autoradiography or fluorescence
Start at bottom and then read upwards 5'-3' to find the sequence of the DNA fragment.
How many primers do you use in Sanger/dideoxy sequencing?
Only use one primer or else you dont know which bands are which to determine the sequence becuase you have two bands migrating the same distance on the gel
What are all the ingredients for Sanger/dideoxy sequencing?
DNA of interest
DNA primer (oligonucleotide)
Uses fluorescent labels of different colors for each ddNTP
I is one reaction containing all four dNTPs and ddNTPs in one tube and separated by electrophoresis in a single lane
Fluorescent detector records the color of the passing bands and generates a sequence