Molecular Techniques Lecture Sep 29

Question 1

What are the 4 methods of identifying DNA?

Answer 1

Restriction enzyme digestion

Southern Blotting

Sequencing

PCR

Question 2

How does restriction enzyme digestion work?

Answer 2

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

Question 3

After a restriction enzyme is added to DNA, what is done with the material?

Answer 3

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

Question 4

What are the steps of a southern blot?

Answer 4

1. Genomic DNA is digested with restriction enzymes and fragments are separated by size on an agarose gel
2. the DNA is denatured and transferred to a membrane
3. 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.
4. 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
5. SItes of prove binding are visualized by radioactive or fluorescent tag on the probe

Question 5

What is the basis for DNA sequencing?

Answer 5

in vitro DNA replication

Question 6

During the in vivo DNA synthesis of sequencing, what is used to terminate chain extension?

Answer 6

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.

Question 7

What are the steps of DNA sequenceing?

Answer 7

1. Design a primer to the complementary strand you’re interested in
2. initiate synthesis with a mix of dNTPs spiked with a ddNTP, in this case ddATP
3. Repeat for all the nucleotides
4. 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)
5. The ddNTPs can be taged with eithe rradioactivty or fluorescent tags
6. THe order of signals tells us the order of bases

Question 8

What is PCR used for?

Answer 8

to amplify desired DNA sequences

Question 9

Why are two primers required in PCR?

Answer 9

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

Question 10

What two tests do we currently use DNA molecule techniques to diagnose disease?

Answer 10

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

Question 11

How are DNA molecular techniques being adapted for diagnosing trisomy 21 in a safer way?

Answer 11

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

Question 12

What are three techniques to determine the abundance of a certain mRNA?

Answer 12

Northern blotting

Q-RT-PCR

Microarray analysis

Question 13

How does a Northern Blot work?

Answer 13

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

1. All RNAs in a cell are separated by size and transferred to a membrane
2. Short defined piece of DNA or RNA-probe is added which will specifically bind to the target RNA
3. Probe is labelled so the target RNA is marked for visualization

Question 14

How does quantitative Real time PCR work?

Answer 14

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

Question 15

Why must this special version of PCR be used for RNA?

Answer 15

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

Question 16

What is microarray analysis used for?

Answer 16

to determine GLOBAL patterns of mRNA expression and identify individual mRNAs that change in diseased states

Question 17

What is the primary advantage of microarray analysis?

Answer 17

Large numbers of genes can be surveyed at one time

Question 18

What is the array in microarray analysis?

Answer 18

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

Question 19

What are the steps for a microarray?

Answer 19

1. make the array with the DNA oligos
2. isolate the mRNA from the patient sample and a reference sample.
3. Convert mRNA to cDNA with rev. transcfiptase
4. Label the cDNAs in the two samples with a different fluorescent tag
5. Incubate both the patient and the reference samples on the same array chip
6. If the mRNA was present in the sample, the corresponding cDNA will hybridize to the appopriate oligo
7. the hybridization is visualized by fluorescence
8. COmpare the fluorescence to a certain mRNA in the reference compared to the patient sample and look for changes in mRNA corresponding to diasease

Question 20

What does microarray data analysis NOT tell?

Answer 20

it cannot tell which mRNA changes “cause” the disease - it’s only correlational

Question 21

WHat are some clinical examples for using RNA molecular techniques?

Answer 21

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

Question 22

What 2 molecular techniques are used to look at proteins?

Answer 22

ELISA

Western blot

Question 23

WHat is the foudnation of most protein characterization techniques?

Answer 23

using antibodies specific for a protein

Question 24

In techniques that use antibodies for detection of certain proteins, what does a primary antibody recognize? What does a secondary antibody recognize?

Answer 24

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

Question 25

How does an ELISA work for the detection of HIV infection?

Answer 25

1. Recombinant HIV proteins are immobilized on the wells of a 96 well plate
2. the patient’s serum is added
3. if the patient’s serum has antibodies against the HIV proteins, they will bind to the antigen at the bottom of the well plate
4. A secondary antibody is added which will bind to any primary antibody that’s bound to antigen
5. 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

Question 26

What will a western blot identify?

Answer 26

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)

Question 27

What are the steps of a western blot?

Answer 27

1. 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
2. Run the proteins on an SDS-PAGE gel to separate by size
3. Transfer proteins to membrane
4. Incubate membrane with primary antibody specific for target protein
5. Incubate iwth secondary antibody specific for the primary antibody in order to visualize the target protein on the membrane

Question 28

How are antibodies being adapted for cancer treatment?

Answer 28

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

Question 29

How is next generation sequencing (deep sequencing) different from the Sanger sequencing?

Answer 29

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.

Question 30

What two types of RNA are used in RNAi?

What do they do?

Answer 30

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

Question 31

Describe the normal mammalian microRNA pathway.

Answer 31

1. DNA encoding a mrRNA is trascrbed by Pol2
2. the single stranded RNA has regions of complementarity and so folds to make a double stranded hairpin
3. The hairpin is released by cleavage by DROSHA
4. The cleaved hairpin is the pre-miRNA and is exported to the cytoplasm
5. In the cytoplasm, the hairpin is trimmed by dicer to generate a 21bp ds RNA
6. 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
7. THe guide RNA from the miRNA will allow the RISC complex to base pair with the target mRNA
8. THe enzymes in the RISC complex will then inhibit the bound mRNA

Question 32

How can the endogenous RNAi pathways be exploited for therapeutic purposes?

Answer 32

1. synthetic siRNAS or plasmids encoding the short hairpinr RNA are introduced to the cel
2. 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
3. THis targets the RISC complexes tot he specific mRNA that has complimentary base pairing to the short ds RNA that was synthetically introduces
4. the RISC complex inhibits the target mRNA

So these are called siRNA orshRNA but they act like miRNAs