Molecular Techniques And Diagnosis

Question 1

Brief overview of DNA sequencing

Answer 1

Fluorescent stained ddNTPs added to DNA template strand (causes it to terminate, depending on what ddNTPs used in diff places) and DNA polymerase.
Produces lots of fragments of different lengths
Can be denatured by heat and separated used gel electrophoresis

Question 2

Uses of DNA sequencing

Answer 2

DNA :
Mutation
Sequencing the DNA

Question 3

Describe restriction analysis (gene level)

Answer 3

Restriction endonucleases recognise specific DNA sequences and cut the DNA
Leaves sticky ends
Can be reversed or different fragments added by DNA ligase

Question 4

Restriction and gel electrophoresis can be used for….

Answer 4

Investigating :
Size of DNA fragments
Mutations
DNA variation
Gene cloning

Question 5

Describe gene cloning….

Answer 5

Plasmid cut using restriction enzyme and gene of interest added - recombinant DNA
Introduced into bacteria - transformation
Bacteria with recombinant DNA put in environment to multiply

Question 6

What is gel electrophoresis used for ?

Answer 6

Separating dna fragments of different sizes

Question 7

Describe gel electrophoresis

Answer 7

1) solution of diff fragments placed in well at negative anode end
2) charge of anodes encourages DNA to move towards positive end
3) larger fragments move slower
4) reference fragments used

Question 8

3 stages of PCR

Answer 8

1) denaturation - 94-96 degrees
2) renaturation - annealing - 50-65 degrees
3) DNA synthesis 75-80 degrees

Question 9

Enzyme in PCR

Answer 9

Thermostable taq DNA polymerase

Question 10

PCR is used to

Answer 10

Amplify specific DNA sequence
Investigate small base mutations
Investigate small deletions or insertions

Question 11

3 things used in allele specific test…

Answer 11

PCR
Restriction analysis
DNA hybridisation

Question 12

Southern blotting is used in DNA to..

Answer 12

Investigate :
Gene structure
Gene expansions
Variations

Question 13

Blotting methods for proteins and RNA

Answer 13

Western blotting - proteins

Northern blotting - RNA

Question 14

SDS page seperates proteins based on…..

Answer 14

Molecular weight

Question 15

SDS page works by…

Answer 15

Detergent SDS denatures proteins and gives them a negative charge proportional to molecular weight
Largest molecular weight moves further

Question 16

Isoelectric focusing seperates proteins based on

Answer 16

pI

Question 17

Isoelectric focusing works by…..

Answer 17

Applying protein to gel with a pH gradient, protein migrates until pI = pH as has no overall charge

Question 18

Difference between polyclonal and monoclonal antibodies?

Answer 18

Polyclonal are produced by lots of B lymphocytes, have multiple epitopes, multiple diff antibodies and specific to one antigen
Monoclonal are produced by 1 B lymphocyte, 1 identical antibody, 1 antigen and 1 epitope

Question 19

Enzyme assays are used to?

Answer 19

Used as a diagnostic tool for metabolic diseases where enzymes are at lower levels

Question 20

What is karyotyping?

Answer 20

Karyotype is a picture of the full set of stained metaphase chromosomes of an individual organised according to chromosome number

Question 21

What is FISH?

Answer 21

Investigation of specific DNA sequences in chromosomes within the cell.

Question 22

What is often used for chromosome investigation?

Answer 22

Chromosome painting

Question 23

What can array comparative genomic hybridisation be used for

Answer 23

Sub microscopic chromosomal deletions / duplications

Gene expression