DNA and RNA analysis

Question 1

what are several methods of analysing DNA?

Answer 1

1. DNA visiualisaiton; agarose gel electrophoresis
2. Amplification; PCR
3. Sequencing; Sanger and Next Generation

Question 2

what is karyotyping?

Answer 2

the process of preparing chromosomes for analysis.

Question 3

what is a karyotype?

Answer 3

number and appearance of chromosomes in the nucleus of a eukaryotic cell.

Question 4

what does chromatin look like under analysis?

Answer 4

heterochromatin; dark > inactive
euchromatin; light > active

Question 5

what is FISH?

Answer 5

fluorescence insitu hybridisation
- staining technique where known sequences of DNA are fluorescently labelled and hybridised to chromosomes

Question 6

what does in situ refer to?

Answer 6

the fact that the fluorescent DNA probe is hybridised to DNA of interphase nuclei or metaphase chromosomes which have been fixed on a slide

Question 7

what does FISH provide researchers with?

Answer 7

a way to visualise and map the genetic material in an individuals cells, including specific genes or portions of genes.

Question 8

what can FISH only do?

Answer 8

detect deletions or duplications of regions specifically targetted by the probe used.

Question 9

PCR

Answer 9

• exponential amplification of a DNA fragment
• usually of known sequence

Question 10

what are the components used in PCR?

Answer 10

• template; DNA to amplify
• primers; short pieces of ssDNA
• polymerase; thermostable enzyme (Taq)
• nucleotides; single base mixture (dNTPs)
• buffer; to maintain pH
• MgCl2; essential for polymerase activity.

Question 11

principles of PCR

Answer 11

• requires incubating at three different temperatures
  = denaturing, annealing, extension

Question 12

background of Sanger sequencing

Answer 12

• invented by Frederic Sanger and co in 1977
• also called dideoxy sequencing because it modified nucleotides called dideoxynucleotides

Question 13

what is process of Sanger sequencing?

Answer 13

1. DNA extracted from the cells of the organism being studied
2. sequencing reaction the performed on the DNA and the sequenced DNA strands are sorted by size using capillary electrophoresis.
3. finally DNA code is read by a computer which displays the data for scientists to use.

Question 14

summary of what sanger sequencing is

Answer 14

A PCR containing fluorescent, chain terminating dideoxynucleotides triphosphates

Question 15

why are fluorescent in sanger sequencing called chain terminating?

Answer 15

because they contain an alteration in the structure that doesn’t allow polymerase to continue sequencing.
- ddTTP, ddATP, ddGTP, ddCTP

Question 16

what are the main causes of cellular dysfunction that underlies different disease states?

Answer 16

disturbances in gene expression as a result of pertrubed transcription or posttranscriptional regulation

Question 17

what is qRT-PCR used for?

Answer 17

for gene expression analysis
- generate cDNA from mRNA, then PCR

Question 18

what is microarray used for?

Answer 18

detect the expression of thousands of genes at the same time

Question 19

what is RNA-seq used for?

Answer 19

allows for full sequencing of the whole transcriptome

Question 20

central dogma of molec. biology

Answer 20

DNA > RNA > protein

Question 21

what is reverse transcriptase-PCR?

Answer 21

generation of cDNA from RNA that will be amplified and run on agarose gel to look at expression of gene of interest in different tissues
- assesses transcript expression

Question 22

what is a housekeeping gene?

Answer 22

gene that’s expression will not be affected by anything done to the tissue or cell.

Question 23

steps of qPCR

Answer 23

1st step; reverse transcription (RT)
2nd step; PCR using SYBR green fluorescent; can bind to ds DNA , unbound will not emit fluorescence
3rd step; fluorescence measured

Question 24

how are results taken in from qPCR?

Answer 24

• output measured in real time in the thermal cycler
• number of cycles taken to reach the threshold value is determined
• proportional to the amount of starting cDNA (mRNA)

Question 25

summary of DNA and RNA analysis

Answer 25

• we can visualise and separate DNA fragments based on the size with the agarose gel electrophoresis
• karyotyping and FISH can visualise and map genetic material and detect gene deletion or duplications
• PCR is used for DNA amplification and for the Sanger/Dideoxy sequencing
• can quantify differences in mRNA expression with qPCR. If a particular seq is abundant in the sample, amplification if observed in earlier cycles; if sequence is scarce, ampification observed in later cycles.