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