Molecular Techniques Lecture Sep 29 Flashcards
What are the 4 methods of identifying DNA?
Restriction enzyme digestion
Southern Blotting
Sequencing
PCR
How does restriction enzyme digestion work?
Restriction enzymes will create double stranded breaks in specific sequen es, thus creating a characteristic set of fragments which can e used to identify pieces of DNA
It can also be used to recombine DNA now
After a restriction enzyme is added to DNA, what is done with the material?
It’s run on an agarose gel.
the small pore size impedes migration of large fragments more than the small fragments, so the larger restruction feagments will get stuck closer to the top.
this separates out the fragments by size and they’re visualized with a DNA dye
What are the steps of a southern blot?
- Genomic DNA is digested with restriction enzymes and fragments are separated by size on an agarose gel
- the DNA is denatured and transferred to a membrane
- The membrane is incubated with a probe, which is a defined piece of DNA that is homologous to the target and labelled with radioactivity or fluorescence for detection.
- If the DNA sequence encoded in the probe is present in the DNA misture, then complenetary strands of probe and target DNA will bind each other
- SItes of prove binding are visualized by radioactive or fluorescent tag on the probe
What is the basis for DNA sequencing?
in vitro DNA replication
During the in vivo DNA synthesis of sequencing, what is used to terminate chain extension?
Dideonucleotide chain terminatos.
DNA synthesis depends on the 3’ OH of the final nucleotide, but ddNTPs don’t have that OH group, so elongation is terminated.
Since they will only terminate at sites with complementary base pairing, the length of a terminated fragment tells the position of the base in a newly synthesized strand.
What are the steps of DNA sequenceing?
- Design a primer to the complementary strand you’re interested in
- initiate synthesis with a mix of dNTPs spiked with a ddNTP, in this case ddATP
- Repeat for all the nucleotides
- ANalyze the fragments on a gel. The size of the fragments correspond to the position of the ddNTP in the sequence (with the smallest fragment having the ddNTP closest to the primer)
- The ddNTPs can be taged with eithe rradioactivty or fluorescent tags
- THe order of signals tells us the order of bases
What is PCR used for?
to amplify desired DNA sequences
Why are two primers required in PCR?
There needs to be a primer for each of the DNA strands. THis is what allows for exponential growth - both strands can be used as templates
What two tests do we currently use DNA molecule techniques to diagnose disease?
Diagnosing HPV: we use RNA probes to hybridize viral DNA and visualize
Identifying BRCA mutations that cause hereditary disease and risk factors through DNA sequencing
How are DNA molecular techniques being adapted for diagnosing trisomy 21 in a safer way?
THey’re developing a PCR analysis for chromosomes 21 in fetal DNA that is free floating in the plasma
the ratio of chroomsomes 21 should be equal to that of another chromosomes (12 is the one usually used for comparison). If the 21:12 raio is actually 1.15 or higher, you can diagnose with downs.
this means you don’t have to do the dangerous amniocentesis
the PCR is required because free floating fetal DNA has a very low concentration and wouldn’t provide good results without amplification first
What are three techniques to determine the abundance of a certain mRNA?
Northern blotting
Q-RT-PCR
Microarray analysis
How does a Northern Blot work?
It’s the RNA equivalent of Southern blotting….
It’s based on DNA-RNA hybridization through complementary base pairing and it’s used to identify one target RNA in a complex misture of al the RNAs expressed in a cell
- All RNAs in a cell are separated by size and transferred to a membrane
- Short defined piece of DNA or RNA-probe is added which will specifically bind to the target RNA
- Probe is labelled so the target RNA is marked for visualization
How does quantitative Real time PCR work?
All the mRNAs in a cell are ocnverted to cDNA by reverse transcriptase
the cDNA derived from a specific mRNA is then amplified using specific primers and PCR
Fluorescent dye is incorporated as the new DNA is synthesized
the amplification will be proportional to the initial number of mRNAs and the intensity fof the fluorescence is therefore proportional to the initial number of copies of the target mRNA
Why must this special version of PCR be used for RNA?
because RNA is not a template for most DNA plymerases and because RNA is single stranded and therefore can’t be amplified exponentially.
THe reverse transcriptase IS a polymerase that will use RNA as a template to form DNA
the cDNA can then be used as a template for normal PCR
What is microarray analysis used for?
to determine GLOBAL patterns of mRNA expression and identify individual mRNAs that change in diseased states
What is the primary advantage of microarray analysis?
Large numbers of genes can be surveyed at one time
What is the array in microarray analysis?
It’s an array of DNA oligos (25 bases long) arranged on a chip.
the oligos match sequence of the mRNA of each gene to be surveyed
they are derived from genes to be analyzed
there can be up to 40,000 genes per chip
What are the steps for a microarray?
- make the array with the DNA oligos
- isolate the mRNA from the patient sample and a reference sample.
- Convert mRNA to cDNA with rev. transcfiptase
- Label the cDNAs in the two samples with a different fluorescent tag
- Incubate both the patient and the reference samples on the same array chip
- If the mRNA was present in the sample, the corresponding cDNA will hybridize to the appopriate oligo
- the hybridization is visualized by fluorescence
- COmpare the fluorescence to a certain mRNA in the reference compared to the patient sample and look for changes in mRNA corresponding to diasease
What does microarray data analysis NOT tell?
it cannot tell which mRNA changes “cause” the disease - it’s only correlational
WHat are some clinical examples for using RNA molecular techniques?
Use RT-PCR with or without sequencing to identify genetic alterations affecting protein coding regions, like OTC and fusion genes in leukemias and lymphomas.
Also use this to screen blood pool for HIV
Use microarraay analysis to look for ONcotype DX - determine risk or recurrence in breast cancer
What 2 molecular techniques are used to look at proteins?
ELISA
Western blot
WHat is the foudnation of most protein characterization techniques?
using antibodies specific for a protein
In techniques that use antibodies for detection of certain proteins, what does a primary antibody recognize? What does a secondary antibody recognize?
a primary antibody will recognize specific target proteins
a secondary antibody will recognize the primary antibody
the secondary antibody will have a tag that emits signal when activated
How does an ELISA work for the detection of HIV infection?
- Recombinant HIV proteins are immobilized on the wells of a 96 well plate
- the patient’s serum is added
- if the patient’s serum has antibodies against the HIV proteins, they will bind to the antigen at the bottom of the well plate
- A secondary antibody is added which will bind to any primary antibody that’s bound to antigen
- the secondary antibody has an enzyme tag, so if you add the substrate fo rthe enzyme a color change will occur if the test is positive
What will a western blot identify?
a specific protein in a comples mix, such as a total cell lysate
it uses two things: the size fractionation of the protein mixture and specific binding of the antibody to the target protein (with secondary antibody for detection)
What are the steps of a western blot?
- prepare the protein by denaturing and incubating with SDS to give a uniform mass:charge ratio so the migration depends on MASS not native charge or shape
- Run the proteins on an SDS-PAGE gel to separate by size
- Transfer proteins to membrane
- Incubate membrane with primary antibody specific for target protein
- Incubate iwth secondary antibody specific for the primary antibody in order to visualize the target protein on the membrane
How are antibodies being adapted for cancer treatment?
Many cancers express very unique proteins, so antibodies to these proteins can be used to target the cancer.
HER2 is an example - it’s a receptor for epidermal GF that is overexpressed in 25% of breast cancers
Herceptin/trastuzamab is an antibody to the HER2 receptor and has become a very effective treatment for breast cancers with overexpression of HER2
How is next generation sequencing (deep sequencing) different from the Sanger sequencing?
It sequences all the DNA in a genome at once by first breaking it up into smaller fragments and sequencing all the fragments at the same time. Then the computer aligns all the fragments to a template.
What two types of RNA are used in RNAi?
What do they do?
They are both small non-coding RNAs that regulate gene expression in the cell: microRNA and short interference RNA (siRNA)
They will target specifi mRNAs and either block their translation or result in their degradation
Describe the normal mammalian microRNA pathway.
- DNA encoding a mrRNA is trascrbed by Pol2
- the single stranded RNA has regions of complementarity and so folds to make a double stranded hairpin
- The hairpin is released by cleavage by DROSHA
- The cleaved hairpin is the pre-miRNA and is exported to the cytoplasm
- In the cytoplasm, the hairpin is trimmed by dicer to generate a 21bp ds RNA
- This ds RNA is then unwound and one strand called the guide strand will be incoporated into a RISC complex, which is an effector complex that contains enzyes that cleave or inhibit mRNA
- THe guide RNA from the miRNA will allow the RISC complex to base pair with the target mRNA
- THe enzymes in the RISC complex will then inhibit the bound mRNA
How can the endogenous RNAi pathways be exploited for therapeutic purposes?
- synthetic siRNAS or plasmids encoding the short hairpinr RNA are introduced to the cel
- THse will enter the endogenous RNAi pathway and be processed to short single stranded guide RNAs, which will then be incportated into the RISC complex
- THis targets the RISC complexes tot he specific mRNA that has complimentary base pairing to the short ds RNA that was synthetically introduces
- the RISC complex inhibits the target mRNA
So these are called siRNA orshRNA but they act like miRNAs
