Gene Principles

Question 1

How does gene expression change?

Answer 1

during development, physiological conditions and disease.

Question 2

Where can we get the primary structure of the protein from?

Answer 2

primary structure of the control region as they regulate how the coding structures work and are not actually transcribed themselves

Question 3

What is Nucleic Acid Hybridization?

Answer 3

Two complementary single-stranded DNA and/or RNA molecules bond together to form a double-stranded molecule.

Question 4

Nucleic Acid Hybridization: what do probes bind to and what does this do?

Answer 4

a specific sequence of RNA and will be seen after the addition of a radioactive or non-radioactive label.

Question 5

why use Nucleic Acid Hybridization?

Answer 5

to assess the composition, physical distribution, abundance and/or relatedness of nucleotide sequences to DNA or RNA

Question 6

Why do we use northern blots?

Answer 6

analysis method used to study RNA. Specifically, purified RNA fragments from a biological sample and can tell size of RNA

Question 7

How do you start the northern blotting technique?

Answer 7

Use an agarose gel and as the phosphate backbone is negative it will move towards the positive area of the gel.

Question 8

What size peices move faster, smaller or bigger?

Answer 8

smaller

Question 9

What is the second step of a northern blotting technique?

Answer 9

You will then blot this onto a nylon membrane by capillary flow – the RNA will stick to this membrane.

Question 10

What is the third step of the northern blotting technique?

Answer 10

You then do your hybridization to see where the probe is sitting.

Question 11

How do you then find you sequence of interest on a northern blot?

Answer 11

You can then add an x-ray film and where the radioactivity appears this is where your probe is

Question 12

What is in situ hybridization?

Answer 12

Can provide information on cell type specific gene expression in a tissue

Question 13

In in situ hybridization is there a nylon membrane used?

Answer 13

No its tissue

Question 14

Can you do Nucleic Acid Hybridizationin different colours?

Answer 14

Yes, makes it easier to visualise.

Question 15

Can you see more than one gene at a time in in-situ?

Answer 15

yes if you colour them,

Question 16

What does the PCR do?

Answer 16

Finds the presence of the transcript – if you can amplify it then you can tell the DNA gene is being expressed – the transcript is there.

Question 17

What is the PCR probe?

Answer 17

Nucleic acids which is used as a short prime site.

Question 18

What is TR-PCR?

Answer 18

real time PCR measure at different points to see whats being transcribed at different points of reaction

Question 19

Can PCR be detected by fluorescence?

Answer 19

PCR is very sensitive and can be detected by fluorescence

Question 20

What is laser capture microdisection?

Answer 20

can amplify small materials and knock out specific information from one tissue to compare to another

Question 21

What is a micro-array?

Answer 21

Label all the transcripts instead of sequence specific probes. • Then simultaneously analyze a large number of immobilized test sequences – “probes”

Question 22

What is In situ oligonucleotide synthesis?

Answer 22

sequence of DNA placed on a chip for analysis

Question 23

What did In situ oligonucleotide synthesis help do?

Answer 23

Different lymphoma cell lines could be outlined. Some people had good results on chemotherapy some had none at all which made it crucial that people knew why and who they could give the chemotherapy to. This can also help chemotherapy be focused

Question 24

What is rna sequencing and sanger sequencing?

Answer 24

Parallel sequence determination, instead of determinng 100s of bases from a single DNA target it can sequence from many DNA targets.

Question 25

How do you know how many of a certain type of molecule is available?

Answer 25

You need to count how many times a sequence appears to identify how many of a certain type of molecule is available.

Question 26

Northern blots, in situ hybridizations and PCR follow the transcription of what?

Answer 26

a small handful of genes.

Question 27

Micro arrays follow the transcription of what via what pattern?

Answer 27

10,000s of genes. Patterns of transcription rather than the activity of individual genes.

Question 28

What does RNa seq offers the potential to sequence what?

Answer 28

whole mRNA population

Question 29

What is the Pro of in situ methods?

Answer 29

good cell and tissue specificity,

Question 30

What are the 4 cons of in situ methods?

Answer 30

not particularly sensitive, quite difficult to quantify, only analyze one or two gene products at a time and are technically difficult.

Question 31

What is the pro of Quantitative Real Time Polymerase Chain Reaction, “qRT-PCR”?

Answer 31

sensitive, can analyze more than one gene product at a time.