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Southern analysis

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

1

Northern analysis

Uses a DNA probe to detect RNAs separated by gel electrophoresis

Tissue specific expression, gene regulation

2

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

3

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

4

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

5

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)

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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

7

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

8

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

9

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

10

Shotgun sequencing

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.

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Madam-Gilbert sequencing

Based on chemical degradation of DNA

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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

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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

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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.


15

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

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What are all the ingredients for Sanger/dideoxy sequencing?

DNA of interest

DNA polymerase

DNA primer (oligonucleotide)

Salt buffer

dNTPs

ddNTPs

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Automated sequencing

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

18

Fluorescence sequence printout

Chromatogram based on chain termination