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Nucleic Acid Techniques

Nucleic acid techniques are useful for gene sequencing, biomarker identification, and synthetic gene creation. Common applications include pathogen identification, gene function studies using transgenic/knockout models, and gene therapy using RNAi. DNA is extracted from cells and purified, then can be amplified via PCR or cloned into vectors for further analysis. Techniques like agarose gel electrophoresis, PCR, sequencing, and mouse transgenic/knockout models allow manipulation and study of genes.

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41 views32 pages

Nucleic Acid Techniques

Nucleic acid techniques are useful for gene sequencing, biomarker identification, and synthetic gene creation. Common applications include pathogen identification, gene function studies using transgenic/knockout models, and gene therapy using RNAi. DNA is extracted from cells and purified, then can be amplified via PCR or cloned into vectors for further analysis. Techniques like agarose gel electrophoresis, PCR, sequencing, and mouse transgenic/knockout models allow manipulation and study of genes.

Uploaded by

sasabugan
Copyright
© Attribution Non-Commercial (BY-NC)
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Nucleic Acid Techniques

EMVB | HLY

Why use nucleic acid?


Gene sequencing is much easier (and cheaper) than protein sequencing Gene biomarkers are better platforms than antibodies (change target = change sequence) Synthetic genes easier to make (PCR!) than synthetic proteins [in vitro studies]

Where gene studies are used


Sequence identify pathogen strain (CHIKV vs DENV), epidemiological significance (DENV strain), Transgenic/gene knockouts/knockins loss of function/gain of function studies to know the function of the gene, understand mechanisms of diseases (and normal processes) RNAi (miRNA/siRNA) gene therapy

DNA extraction, isolation, purification


Most often done by kits (optimized) Crude extraction: lyse cell UV detection 260nm pure DNA is conventionally and usually defined if A260/A280 is 1.7 2.0

Agarose gel electrophoresis


Similar to PAGE Instead of polyacrylamide, agarose is used Allows for larger molecules to pass through DNA stain or chelating agent (EtBr) can be used to visualize DNA bands

Amplification techniques
cloning into BACs or YACs (depending on size of the gene) PCR

Cloning (BAC)
BAC/YAC/MAC depending on size of gene of interest Distinguish: incorporated vector (what you want) from no vector/no gene of interest

ACTIVITY 1
Generate your own GMO!

PCR
dNTPs + primer + template + Taq polymerase Thermal cycler: 95 (denature DNA) 55 (anneal primer) 72 (polymerize)

Designing primers for PCR


Trickiest part: primer design
Tm around 50-60o (check AT and GC content) Forward and reverse primer same Tm Specificity Minimal / no hairpin formation No Homo / heterodimer (of primer(s)) formation

http://eu.idtdna.com/scitools/scitools.aspx

Quantification (of the PCR product)


Conventional: RT-PCR (or qPCR)

Application (amplification + AGE)


RFLP (Random fragment length polymorphism) Paternity testing Disease carrier status For the non-translated regions: Susceptibility to diseases / drug resistance Transplant compatibility

Whos the daddy?

Variable number tandem repeats VNTRs


Aside from random fragmentation patterns, you can look at VNTRs Highly variable, so same VNTR = more likely to be related

ACTIVITY 2
The alleged switch

Sequencing techniques
Sanger dideoxy method Next generation sequencing

Sanger dideoxy method


2, 3 dideoxy nucleotides prevent further polymerization Gel visualization dNTPs, template DNA, polymerase + specific ddNTP per well in the gel

Next generation sequencing: Sequence by synthesis


[illumina video]

Gene Manipulation (mouse models)


Transgenic mice Knockout mice selective gene is removed

Creating mouse models


target

gene

microinjection

targeting vector

neor

TK

homologous recombination

gene

neor

transgenic

knockout

Generating transgenic mice


random integration of exogenous DNA occurs frequently 1030% of injected eggs result in a transgenic offspring. Multiple copies are inserted in a tandem fashion

Generating knockout mice


homologous recombination: recombination between the exogenous DNA and its homologous chromosomal site in the embryonic stem (ES) cells

target

ES Cells

neor

TK

Screening to get the knockout ES cells only both positive and negative screening needed

Recombinants with random insertion

Selection for neor positive

Selection for TK negative

ES cells with homologous recombination

Knockout vs transgenic models


Knock-out/in
Cells ES cells

Transgenic
fertilized eggs

Homologous DNA
Insertion site

yes
targeted

no
random

Copy number

1
usu. Loss-of-function

tandem repeats
usu. Gain-of-function no

Reproducibility*

yes

ACTIVITY 3 (take home)


Transgenic fly http://www.hhmi.org/biointeractive/vlabs/transg enic_fly/index.html

Controlling translation -- RNAi


General rule: no double stranded RNA in cytoplasm [video]

Challenges in using DNA


DNA does not tell you the protein (introns, 3 and 5 untranslated regions (UTRs)
Usually, cDNA is used but mRNAs are difficult to handle Bioinformatics tools can help (identify ORFs, and BLAST)

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