Session 12-Intro To Molecular Techniques Flashcards Preview

Semester 1-MCBG > Session 12-Intro To Molecular Techniques > Flashcards

Flashcards in Session 12-Intro To Molecular Techniques Deck (38):
1

What are the five ways in which DNA can be analysed at a gene level?

Restriction enzymes
DNA gel electrophoresis
PCR variations
Southern blotting
Microarrays

2

What are the three ways in which proteins can be analysed?

Protein electrophoresis
Immunoassays
Enzyme assays

3

How is DNA analysed at a nucleotide level?

DNA sequencing

4

What are the two ways in which DNA can be analysed at a chromosome level?

Karyotyping
FISH

5

Why do bacteria produce endonucleases?

To recognise and degrade foreign DNA

6

Which restriction site does the restriction enzyme EcoRI recognise?

GAATTC

7

How does DNA gel electrophoresis separate fragments?

Depending on their size

8

Why does DNA move towards the positive electrode in gel electrophoresis?

DNA is negatively charged (PO4 3-)

9

What are the requirements for gel electrophoresis? (4)

1. Gel
2. Buffer
3. Power supply
4. Stain/detection (fluorescent markers)

10

Why use restriction analysis? (4)

1. To investigate the size of DNA fragments
2. To investigate mutations eg. Sickle cell disease
3. To investigate DNA variation
4. To clone DNA

11

What are plasmids?

Small circular dsDNA found in bacteria which carry genes to replicate independently

12

What are the four basic steps of gene cloning?

1. Isolate relevant gene of interest following digestion with restriction enzymes
2. Insert gene of interest into plasmid vector (recombinant DNA molecule)
3. Introduce recombinant DNA molecule into suitable host cells eg. E. coli
4. Identify and isolate clone containing DNA of interest

13

Why are human genes cloned? (4)

1. To make useful proteins eg. Insulin
2. To find out what genes do
3. Genetic screening
4. Gene therapy

14

What does reverse transcriptase use as a template for DNA?

RNA

15

What is PCR used for?

1. DNA amplification
2. To investigate single base mutations eg. Tay Sachs, Sickle cell
3. To investigate small deletions or insertions eg. Cystic fibrosis
4. To investigate variation, genetic relationships

16

What is Taq polymerase?

DNA polymerase derived from hot springs bacteria- can withstand the high temperature of PCR

17

Describe the process of PCR (5)

1. Amplification of target DNA
2. Thermostable DNA polymerase (Taq)
3. Pair of primers (forward and reverse), uniquely defining the region to be copied
4. Temperature cycles of denature, anneal and polymerise
5. Repeated copying results in exponential increase in DNA

18

On what basis are proteins separated in protein gel electrophoresis?

Size, shape or charge

19

What is the difference between DNA and protein gel electrophoresis?

Protein g.e. Uses a vertical, rather than horizontal, method

20

What is native electrophoresis?

Using the intrinsic properties of the protein to separate it

21

What are the methods of separating proteins on the basis of size called?

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Two-dimensional electrophoresis (2D-PAGE)

22

What happens in SDS-PAGE?

The 3D structure of the protein is broken, the protein is made negative and the disulphide bonds are broken i.e. The secondary and tertiary structure of the protein is gotten rid of so the protein size is known

23

How are proteins separated on the basis of charge?

Isoelectric focusing (IEF)

24

What happens in IEF? (3)

1. A stable pH gradient is established in the gel after application of an electric field
2. Protein solution is added and electric field is reapplied
3. After staining, proteins are shown to be distributed along pH gradient according to their pI values

25

Describe the process of protein identification (proteomics) (4)

1. Digest protein with trypsin
2. Perform mass spectrometry
3. Generate a list of peptide sizes
4. Use database of predicted peptide sizes for known proteins to identify the protein

26

What is the difference between proteomics and molecular diagnosis?

Proteomics = analysis of all proteins expressed from genome
Molecular diagnosis = analysis of a single purified protein

27

Which mnemonic is used for the different types of blots and what they are used for?

SNOW DROP:
S - southern blotting corresponds to D (DNA)
N - northern blotting corresponds to R (RNA)
Os cancel out
W - western blotting corresponds to P (protein)

28

Which method is used to measure the concentration of proteins in a solution?

Enzyme-linked immunoabsorbent assay (ELISA)

29

Describe the steps of ELISA

1. Antigen-coated well
2. Specific antibody binds to antigen
3. Enzyme-linked antibody binds to specific antibody
4. Substrate added and converted by enzyme into coloured product; rate of colour formation is proportional to amount of specific antibody

30

What is the difference between ELISA and radioimmunoassays?

Radioimmunoassay works in the same way as ELISA but uses a radio-labelled primary antibody

31

Name two continuous enzyme assays

Spectrophotometry
Chemoluminescence

32

Name two discontinuous enzyme assays

Radioactivity
Chromatography

33

What are the serum enzyme markers for liver damage/disease?

Aspartate transaminase (AST)
Alanine transaminase (ALT)

34

What is the serum enzyme marker for pancreatitis?

Amylase/lipase

35

What is the serum enzyme marker for bone disorders?

Alkaline phosphatase

36

What is the standard for diagnosis of an MI?

Measurement of cardiac troponin I (cTnI) by ELISA

37

Give an example of when enzymes are used to measure clinically important metabolites

Measurement of glucose concentration with glucose oxidase:
Test strips-H2O2 converted to coloured dye
Glucose monitor-glucose oxidase used as biosensor

38

Why do proteins move in gel electrophoresis?

Proteins are charged molecules and will move towards either the anode or cathode if placed in an electric field