Techniques in Molecular Biology

Question 1

Why use molecular biology techniques?

Answer 1

Understand the genetic basis of disease requires the analysis of DNA

Question 2

how do we target a gene or an exon?

Answer 2

Analyse or manipulate a single human exon or gene

Specifically recognise
a short DNA sequence of interest

Nucleotide hydridisation

Selectively amplify =a short DNA sequence of interest

Use DNA polymerases to make multiple copies of sequence

Question 3

how many hydrogen bonds between a and T

Answer 3

2

Question 4

how many between c and g

Answer 4

3

Question 5

how can we break the h+ bonds in DNA

Answer 5

heating

Question 6

can seperated DNA strands be re assocated again?

Answer 6

yes

Question 7

Hybridisation?

Answer 7

association of any two complementary nucleic acid strands to form a double-stranded nucleic acid

Question 8

what is the purpose of PCR

Answer 8

for amplifying areas of DNA

Question 9

what are the componnets used for PCR

Answer 9

dna
primers
nuceleotides
buffers
dna taq polymerase

Question 10

what is the point of primers in PCR

Answer 10

• single-stranded DNA that help DNA polymerase to initiate the synthesis of new DNA strands.
  Primers are complementary to the target DNA sequence

Question 11

WHAT IS THE POINT OF nucleotides in pcr

Answer 11

They act as raw material for new DNA synthesis

Question 12

what is the point of a buffer in pcr

Answer 12

A salt solution used to stabilize the reaction components, especially the DNA, and maintain an optimal pH during the reaction

Question 13

what is the pupose the enzy,e dna taq poymerase in pcr

Answer 13

The enzyme that synthesizes fresh DNA strands complementary to the target DNA sequence

Question 14

why do we purposely use taq?

Answer 14

enzymeable to withstand the protein-denaturing conditions, required during PCR.

Question 15

what does pcr increase

Answer 15

PCR increases the yield of DNA exponentially

Question 16

what does pcr allow?

Answer 16

allows the in-vitro amplification of specific target DNA sequences using a single copy

Question 17

Detection and identification of the PCR product is usually carried out by:

Answer 17

agarose gel electrophoresis
DNA sequencing

Question 18

summarise Gel electrophoresis:

Answer 18

Separate DNA according to size

Molecules are pushed through a gel that contains small pores by an electric field

One end of gel has a +ve charge and other –ve.

DNA is –vely charged due to the phospate group, will be pulled toward the +ve end of gel.

Smaller molecules migrate more quickly and travel further

Question 19

Visualisation of PCR products

Answer 19

Loading buffer is added to the PCR product and then loaded into wells

A marker is added to one well, ‘ladder’ with known size bands

Visualise DNA – gel is stained with a fluorescent dye that binds to DNA and can see when placed on a UV transilluminator

Size of DNA – imagine horizontal line running from ladder to DNA

Question 20

what can go wrong when doing pcr

Answer 20

Does not amplify

Contamination

Non-specific priming. Primers are not specific to sequence intended and see wrong size product or banding effect

Question 21

how to improve the pcr reaction

Answer 21

Redesign Primers
Increase annealing temperature
Alter number of cycles performed

Question 22

advantages of pcr

Answer 22

Easy to set up (real-time PCR)
Fast turnaround time

Question 23

disadvantages of pcr

Answer 23

Extremely liable to contamination
High degree of operator skill required

Question 24

how is pcr used in pregnancy

Answer 24

For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid

Question 25

what can pcr detect

Answer 25

-direct sequencing of DNA
-restriction enzyme analysis (RFLP)

Question 26

Other advantages of PCR in forensic science are:

Answer 26

relatively simple to perform and simple to standardize
results obtainable within 24 hours.