molecular diagnostic methods

Question 1

what do molecular methods look for

Answer 1

nucleic acids

Question 2

what is a gene

Answer 2

basic unit of hereditary information of an organism
consists of a nucleotide sequence

Question 3

each nucleotide consists of

Answer 3

a phosphate group
sugar
bases - purine = adenine or guanine
- pyrimidine = cytosine or thymine or uracil

Question 4

what is PCR

Answer 4

polymerase chain reaction
an amplification technique for cloning the specific or targeted parts of a DNA sequence to generate thousands to millions of copies of DNA of interest

Question 5

what do you need for PCR

Answer 5

sterile lab conditions
a buffer for the reaction to take place
a mixture of deoxyribonucleotides in equal measures
primers - about 20 bases long, specific for part of genome of target causative agent
heat resistant enzyme - Taq polymerase
positive and negative control

Question 6

what is taq polymerase

Answer 6

a DNA polymerase extracted from the bacteria Thermus aquaticus that lives in thermal springs at 70 degrees

Question 7

what is the positive control

Answer 7

PCR sample in which DNA is added that was extracted from clinical material previously proved to be positive for the specific microorganism, or DNA extracted from pure lab culture

Question 8

what is negative control

Answer 8

a sample in which purified water is added instead of DNA, or DNA extracted from a clinical sample previously proved to be negative

Question 9

what is the machine called

Answer 9

PCR thermocycler

Question 10

3 steps of PCR repeated how many times

Answer 10

denaturation
hybridisation or annealing of primer
elongation
repeated 30-35 times
takes 2 hours and fragment is copied 2n times (n is number of cycles)

Question 11

denaturation

Answer 11

seperation of the double stranded DNA in to 2 single stranded chains as a result of the high temp
93-95 degrees

Question 12

hybridisation/annealing of primer

Answer 12

cooling to 50-60 degrees, depending on composition of primer base
primer binds to its complimentary base sequence on the strands
the primers delimit the target DNA fragment that is amplified in the PCR reaction
forward and reveres

Question 13

elongation

Answer 13

extension of the DNA chain
taq polymerase binds th free dNTPs from the PCR mixture and creates a new chain, which is complimentary to the chain of which it is a copy
at 72 degrees

Question 14

electrophoresis in agarose gel principle

Answer 14

used for visualisation of the DNA amplified in the PCR reaction
PCR product placed in wells, mixed with bromophenol blue dye (stains PCR product and lowering it to the bottom of the well)
PCR product travels through the agarose gel by electric current
the dye only binds to double stranded DNA

Question 15

visualisation of DNA after electrophoresis

Answer 15

we see the DNA as a band on the gel
in 1 well we put a DNA marker - contains part of DNA molecule of precisely known length
chamber with UV source and camera
positive PCR reaction is seen by a signal in the gel
compare signals obtained in the wells of PCR product and marker to define the approximate size of amplified DNA fragment

Question 16

reverse transcription PCR

Answer 16

consists of the reverse transcription of RNA in to complementary cDNA and its amplification by PCR

Question 17

what do we use reverse transcriptase PCR for

Answer 17

to amplify genome of an individual virus

Question 18

real time PCR

Answer 18

follow the creation of the PCR product on the computer in real time
allows us to quantify the amplified part
the new PCR products are marked with fluorescent dye
the quantity of fluorescence is proportional to the quantity of the PCR product produced

Question 19

multiplex PCR

Answer 19

instead of a single pair of primers, 2 or more pairs of primers are used so instead of 1 DNA fragment, several are amplified in the reaction
more difficult to optimise but saves time and chemicals

Question 20

nested PCR

Answer 20

used when we don’t obtain sufficient PCR product to be visible by electrophoresis in the agar gel
2 reactions performed with 2 primers
product of first PCR reaction serves as a mold for the second reaction
the primers used in the second reaction are located inside the fragment amplified in the first PCR reaction

Question 21

RFLP stands for

Answer 21

restriction fragment length polymorphism

Question 22

RFLP method

Answer 22

type of polymorphism that results from variation in the DNA sequence recognised by restriction enzymes
restriction endonucleases are bacterial enzymes used to cut DNA at known locations
to detect variations in homologous DNA molecule sequences
mostly used in forensic cases, parent testing and gene mapping studies

Question 23

RFLP stages

Answer 23

1. extract DNA from sample
2. restriction digestion of a test sample of DNA - using restriction endonucleases from specific cleavage sites in the DNA
3. agarose gel electrophoresis
4. hybridisation (southern blot) and visualisation - DNA transferred to a filter membrane and labelled by specific DNA probes. the length of fragment is determine according to their complementariness with the probe

Question 24

western blotting general

Answer 24

to detect and identify small quantities (less than 5ng) of protein in a sample
homogenised tissue, cells or body fluids
antigen-antibody reaction
high sensitivity

Question 25

western blot 1st stage

Answer 25

1. SDS PAGE - sample is prepared by electrophoresis and then gets SDS detergent and B mercaptoethanol. the protein becomes denatured and transform to primary lines structure with a negative charge
   protein mixture undergoes electrophoresis in polyacrylamide gel - protein move to anodes and divide based on mass

Question 26

western blot 2nd stage

Answer 26

transfer of proteins from gel to membrane
gel covered by PVDF membrane and placed between 2 filter papers and 2 sponges soaked in buffer solution
spontaneous adsorption and electric current separates proteins from gel and transfers them to membrane
success of transfer seen by staining membrane with Ponceau S or Coomassie Brilliant Blue

Question 27

3rd stage of western blot

Answer 27

membrane with proteins is immersed in solution of primary antibodies
then immersed in solution of secondary antibodies (that have a bound enzyme)
incubation and rinsing of membrane in buffer to remove excess secondary antibodies
add chromogenic substrate and precipitate forms at site of antibodies
can add chemiluminescent substrate - a signal is emitted at sites where secondary antibodies are located

Question 28

positive western blot

Answer 28

disintegration of substate and the staining of a line at the site where antibodies reacted (target protein)
molecular mass of target protein in the test in compared with the known size of the protein (protein marker)

Question 29

sampling for molecular methods

Answer 29

material sampled in sterile conditions and sent to lab ASAP
if longer than 1h to lab - some materials eg CSF or tissue have to be at 4–20 degrees
if blot - sample before atb given
indicate which test you want