Advanced molecular techniques

Question 1

What are allele-specific primers?

Answer 1

Refers to primers whose 3’ base sequence is complementary to one allele but not the other

Question 2

What happens if a primer’s 3’ end is complementary to a DNA sequence in PCR?

Answer 2

Taq polymerase can elongate new DNA strand

Question 3

What happens if a primer’s 3’ end is not complementary to a DNA sequence in PCR?

Answer 3

Taq polymerase cannot elongate the new DNA strand

Question 4

What are allele-specific probes?

Answer 4

Refers to probes which are completely complementary to base sequence of one allele but not the other

Question 5

How do allele-specific probes bind to different alleles?

Answer 5

Bind tightly to alleles they are specific to

Bing less tightly to other alleles

Question 6

How would results of Southern blotting using allele-specific probes be interpreted?

Answer 6

Darker band means more allele-specific probe bound

so allele specific to probe is present there

Question 7

What is the first step in reverse transcriptase PCR?

Answer 7

Extract mRNA for wanted gene from the cell

Question 8

What is done to the extracted mRNA in reverse transcriptase PCR?

Answer 8

Add DNA primer to it

which contains lots of T bases

Question 9

What does the DNA primer do with mRNA in reverse transcriptase PCR?

Answer 9

DNA primer containing lots of T bases

binds to polyA tail in mRNA

Question 10

What does reverse transcriptase do the the DNA primer bound to mRNA in reverse transcriptase PCR?

Answer 10

Reverse transcriptase elongates the DNA primer as cDNA

by reading the mRNA

Question 11

What happens to mRNA after the formation of cDNA in reverse transcriptase PCR?

Answer 11

Digested by RNase enzyme

Question 12

What happens to the single-stranded cDNA in reverse-transcriptase PCR?

Answer 12

Undergoes one cycle of PCR
one DNA primer binds
to produce double-stranded cDNA

Question 13

What happens to double-stranded cDNA in reverse-transcriptase PCR?

Answer 13

Undergoes many cycles of PCR
both DNA primers bind
to produce copies of cDNA

Question 14

What does a microarray chip look like?

Answer 14

Slide with grid on it

Question 15

What does each dot on a microarray chip contain?

Answer 15

Single-stranded sections of DNA from one gene only

Question 16

What does microarray conditional gene expression compare?

Answer 16

Compares gene expression of a gene

in normal cell to diseased cells

Question 17

What is the first stage of microarray conditional gene expression?

Answer 17

Reverse transcriptase PCR for normal cell and diseased cell

to produce single-stranded cDNA

Question 18

How is cDNA from the normal cell and diseased cell labelled in microarray conditional gene expression?

Answer 18

Normal cell cDNA is fluorescently labelled green

Diseased cell DNA is fluorescently labelled red

Question 19

What is done to the normal cell cDNA and diseased cell cDNA in microarray conditional gene expression?

Answer 19

Add the cDNA to the relevant gene in the microarray chip

Question 20

How are the results of microarray conditional gene expression interpreted?

Answer 20

Look at colour of dot

• if more green means diseased cell has reduced gene expression for that gene
• if more red means diseases cell has increased gene expression for that gene
• if yellow means equal expression in each cell

Question 21

What does array comparative genome hybdridisation compare?

Answer 21

Compares genes in normal cell to genes in diseased cell

Question 22

What is the first stage of aCGH?

Answer 22

Extract all DNA from normal cell and from diseased cell

Question 23

What is done to the DNA extracted from the normal cell and the diseased cell in aCGH?

Answer 23

Digest with restriction enzymes

Fluorescently labelled

• normal cell is green
• diseased cell is red

Question 24

What is done to the fluorescently labelled DNA fragments in aCGH?

Answer 24

Added to the microarray chip

Question 25

How are the results of aCGH interpreted?

Answer 25

Look at colour of dot for each gene

• if more green, means diseased cell has deletions
• if more red, means diseased cell has duplications
• if yellow, means normal

Question 26

Are minisatellites in coding DNA or non-coding?

Answer 26

Non-coding

Question 27

What are minisatellites?

Answer 27

Repeating base sequences

Question 28

How specific are minisatellites to an individual? Why?

Answer 28

Unique to individuals

but somewhat resemble minisatellites of parents, since inherited DNA from parents

Question 29

What are the processes involved in DNA fingerprinting?

Answer 29

Restriction analysis

followed by Southern blotting

Question 30

What are the probes complementary to in DNA fingerprinting?

Answer 30

Complementary to minisatellites in DNA

Question 31

How are the results of DNA fingerprinting interpreted?

Answer 31

Compare banding patterns

• somewhat similar between parent and child
• exactly similar if of same person

Question 32

What are the uses of DNA fingerprinting?

Answer 32

Forensics - suspects to site of crime

Paternity testing

Question 33

What is the first stage in Fluorescent In Situ Hybridisation (FISH)?

Answer 33

Add fluorescently labelled probes to DNA

Question 34

What do the fluorescently labelled probes in FISH do?

Answer 34

Bind to complementary base sequences in DNA

Question 35

How are the results of FISH interpreted?

Answer 35

Look at fluorescently labelled probe under microscope

where it had bound to DNA

Question 36

What is the use of FISH?

Answer 36

Detects mutations

Question 37

What is the first stage of whole chromosome pointing?

Answer 37

Add fluorescently labelled probes to all of chromosome length

Question 38

How are the results of whole chromosome painting interpreted?

Answer 38

Look at colour of entire chromosome

Question 39

What are the uses of whole chromosome painting?

Answer 39

Detect numerical abnormalities

Detect structural abnormalities

Question 40

How are the results of karyotyping interpreted?

Answer 40

Look at banding pattern of chromosomes formed by staining