Molecular Techniques

Question 1

How do restriction enzymes work?

Answer 1

specific endonucleases recognise and cut palindromic recognition sequences

Question 2

How does gel electrophoresis work?

Answer 2

gel runs from negative electrode to positive, dna is negatively charged so it runs up the gel

smaller fragments of DNA move further than larger fragments

restriction endonucleases cut dna into fragments of varying size

Question 3

When is restriction analysis appropriate?

Answer 3

to investigate the size of DNA fragments (e.g. small deletions)

to investigate mutations (sickle cell disease)

to investigate dna variation (dna fingerprinting)

to clone DNA

Question 4

How does gene cloning work?

Answer 4

gene of interest isolated using restriction enzyme

gene of interest inserted into plasmid vector (recombinant DNA)

recombinant DNA inserted into suitable host cell

identify and isolate the clone containing the DNA of interest

Question 5

How is gene cloning useful?

Answer 5

finding out what genes do
genetic screening e.g. Huntington’s, Cystic Fibrosis
gene therapy

Question 6

What things does PCR require?

Answer 6

heat stable taq polymerase

pair of primers (forward and backward), uniquely defining region to be copied

Question 7

How does PCR work?

Answer 7

1) Denaturation (95c)
2) Primers added
3) Primer extension (72c/Taq Polymerase)

Question 8

When is PCR useful?

Answer 8

investigating single base mutations
investigating small deletions or insertions
investigating variation, genetic relationships e.g DNA profiling

Question 9

How does protein gel electrophoresis work?

Answer 9

proteins are charged molecules and will move towards the anode or cathode if placed in an electric fluid

proteins can be separated on the basis of size, shape or charge

Question 10

How does SDS page work? (form of protein gel electrophoresis)

Answer 10

separation of proteins on the basis of size

detergent breaks disulphide binds so you examine the primary structure only

Question 11

How does isoelectric focusing work? (form of protein gel electrophoresis)

Answer 11

proteins separate on the basis of charge by running through a gel which separates on basis of pH

Question 12

How does 2D-PAGE work?

Answer 12

• allows separation of complex mix of proteins

- e.g pH and size at the same time

Question 13

What is proteomics?

Answer 13

digesting protein with trypsin and performing mass spectrometry to work out size. then, comparing against a database of peptide sizes.

Question 14

What is Western Blotting?

Answer 14

Artificial antibodies are created that react with a specific target protein. The sample to be tested is prepared and put together with these antibodies on a membrane – if the specific protein sought for is present, after a gel electrophoresis step this will result in an accordingly stained band on the western blot

Question 15

How do enzyme linked immunoabsorbant assays work?

Answer 15

Can be used to measure the concentration of proteins in solution e.g. hormones

• antigens in well
• specific antibodies added
• enzyme linked antibodies added which binds to primary antibodies
• substare added, rate of colour formation equivalent to amount of specific antibody

Question 16

What do enzyme assays do?

Answer 16

measure the amount of product

Question 17

What are some clinically important markers of disease (enzymes) in the body?

Answer 17

Alanine transaminase (ALT)/Aspartate transaminase (AST)-Markers for liver damage / disease

Amylase/ lipase- Marker for pancreatitis

g-glutamyl transferase- Marker for liver damage Increased by alcohol

Alkaline phosphatase- Marker for bone disorders

Question 18

How does DNA hybridisation work?

Answer 18

Denaturing DNA
adding fluorescent probe
and reannealing

Question 19

How does southern blotting work?

Answer 19

digest dna with restriction enzymes
separate dna fragments by gel electrophoresis
transfer single stranded dna fragments to nylon
hybridise filter with labelled gene probe
detect hybridisation (and hence dna of interest) via exposure filter to x-ray film

Question 20

When is southern blotting useful?

Answer 20

Southern blotting allows us detect pieces of DNA from complex mixtures that would otherwise be very difficult to detect.

good for large deletions or duplications

It also allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.

In many cases we can use Southern blotting in combination with PCR to detect things like gene structure, gene expansions and repeats, mutations and genetic variation etc. (allele specific probes)

Question 21

How does DNA sequencing work?

Answer 21

by adding dideoxy nucleotides to DNA fragments, the chain terminates at different places. DNA fragments of different sizes produces. By using a gel electrophoresis, a sequence can be read from top to bottom.

Question 22

How does RT-PCR work?

Answer 22

gene expression measured (mRNA examined)

RNA template converted into DNA and then amplified

Question 23

How does microarray technology work?

Answer 23

analysis on a genome wide level, checks gene expression (red vs green)

good for comparison, works via hybridisation principles

Question 24

How does array competitive genome hybridisation work?

Answer 24

checks for micro deletions and duplications by comparisson of sample DNA to normal dna

Question 25

How does DNA fingerprinting work?

Answer 25

examines non coding areas of the genome, by examine micro satellites (repeated areas of 30-60 bases)
Restriction enzymes and southern blotting with probes for micro satellites can show genetic links.

Question 26

What is FISH?

Answer 26

denatured DNA labelled with fluorescent dye and hybridise. It highlights regions within DNA via labelled probes (chromosome painting, can show translocations)