Genetics - Gene Expression Analysis

Question 1

What is reverse transcriptase analysis based on?

Answer 1

Some organisms contain a high abundance of a specicif mRNA like chicken oviducts ovalbumin

Question 2

Reverse Transcriptase

Answer 2

An enzyme transcribing ssRNA into DNA, able to synthesised a double helix DNA once the RNA has been reverse transcribed

Question 3

Complementary cDNA

Answer 3

A DNA synthesised from an ssRNA template catalysed by reverse transcriptase

Question 4

How is DNA cloned with RT?

Answer 4

Uses ssRNA to synthsise a complementary strand of DNA

Question 5

What is the stem loop structure in RT?

Answer 5

Where the DNA folds back on itself at the 3’ end, consisting of a short dsDNA unit

Question 6

What is the advantage of cDNA?

Answer 6

Does not contain the introns foudn in genomic DNA

Question 7

What is done with cDNA?

Answer 7

Amplified by PCR then Joined to a vector.

Question 8

RT-PCR

Answer 8

This is conversion of RNA to cDNA by RT with then PCR of the cDNA

Question 9

What can RT-PCR be used for?

Answer 9

Comparison of gene expression between samples(different embryonic stages, tissues or same cell under diff ocnditons)

Question 10

What is the first step of ligating cDNA to a vector?

Answer 10

Vector must be cleaved with RE and linearised, creating a site of which cDNA can be inserted.

Question 11

How is the cDNA then generated?

Answer 11

From mRNA using RT, where adaptors are modified to both ends containing specific RE sites.

Question 12

How are complementary overhangs of both vector and cDNA generated?

Answer 12

Using the same restriction endonucleases

Question 13

What is the function of DNA ligase?

Answer 13

Covalently binds DNA strands of 5’ phosphate of one to 3’ hydroxyl of the other.

Question 14

What is phosphodiester bond formation coupled with?

Answer 14

Pyrophosphate hyrdolysis with negative G change.

Question 15

What is the structure of DNA ligase?

Answer 15

NTD recognises DNA subsrate
Central catalytic domain catalyses bond formation
CTD interacts with proteins and cofactors.

Question 16

How is RNA isolated for RT-PCR?

Answer 16

Phenol-chloroform extraction, Column-based RNA purification or magnetic bead-based isolation.

Question 17

What is the process of RT-PCR?

Answer 17

PCR amplified where product analysed by fluoressence or real-time PCR(qPCR)

Question 18

How is RT-PCR gene expression analysed?

Answer 18

If amount of PCR product is lower in experimental group, the gene has lower expression, as amount of RNA transcript is generally correlated with level of gene expression.

Question 19

What is the process of microarray expression analysis?

Answer 19

cDNA generation, labelling with Cy3/Cy5, hybridization with primers on microarray where fluorescence signals intensity analysed.

Question 20

Photolithography

Answer 20

This is where light is used selectively to remove material from a substrate

Question 21

How is a microarray chip generated?

Answer 21

Photolithography.

Question 22

Why is DNA microarray advantageous?

Answer 22

Analysis of expression of thousands of genese at one time using DNA microarray.

Question 23

What is the structure of a DNA microarray chip?

Answer 23

A microscope slide printerd with thousands of slots in defined positions, each containing a known DNA sequence.

Question 24

How is microarray analysis performed?

Answer 24

mRNA collected from a reference(healthy indiviudal) and experimental(a diseased individual)
Both labelled with unique fluorophores after conversion into cDNA
Hybridised with microarray slides where emissivity is assesed.

Question 25

In Situ Hybridization

Answer 25

This is a technique used to detect/localise speciifc NA sequences within intact cells or tissues.

Question 26

What is the first step in ISH?

Answer 26

Probe prepared being a DNA/RNA complementary to target sequence of interest, labbeled.

Question 27

What is the second step in ISH?

Answer 27

Tissue of interest fixated and inserted with the probe, hybridizing within cellls or tissue with complementary sequence.

Question 28

What is the third step in ISH?

Answer 28

Bound probe is detected using fluorescent methods and visualised under confocal microscopes to visualise location of target NA.

Question 29

What can ISH be used for?

Answer 29

Locating where single genes are expressed as well as time and location of the mRNA in the sample.

Question 30

How is ISH visualised?

Answer 30

Auroradiography?

Question 31

Autoradiography

Answer 31

This is the detection/visualisation of radioactive molecules by emitted radiation in contact with photographic fillm.

Question 32

How does Autoradiography work?

Answer 32

Silver halite crystals are excited and destabilised, attracting silver ions from solution forming clusters.

Question 33

What are the clusters in AR proportional to?

Answer 33

Amount of radiation.

Question 34

What does northern blotting use instead?

Answer 34

RNA in formaldehyde in agarose gel in GE with transfer to membrane.

Question 35

Epigenetics

Answer 35

This is the study of heritable changes in genome function not assocaited with DNA sequqence alteraitons.

Question 36

How is epigenetic regulation determined?

Answer 36

3rd Generation Seqeuncing

Question 37

Why is Epigenetic modificatio nimportant to determine?

Answer 37

A bacteria might gain advantages by epigenetic regulation isntead of genetic.