Molecular Diagnosis Techniques

Question 1

What are the requirements to perform DNA gel electrophoreses.

Answer 1

A gel matrix that allows separation of fragments, a buffer that allows the DNA fragments to move across the gel, a power supply that allows a charge difference to be generated and a stain/or some method of detecting DNA fragments.

Question 2

What is the purpose of DNA gel electrophoresis?

Answer 2

To separate DNA fragments on the basis of size, as smaller molecules move further along the gel than larger molecules.

Question 3

Why would you use restriction anayalsis?

Answer 3

To clone DNA, to investigate DNA variation, to investigate mutations, and to investigate the size of DNa fragments such as in the case if small deletions.

Question 4

What does DNA ligase do?

Answer 4

Acts as the molecular glue and sticks the fragments of DNAback together.

Question 5

What is a plasmid?

Answer 5

Small, circular ds DNA, found in bacteria, mini chromosomes, carry genes to replicate independently, can transfer to different areas and often carry genes for antibiotic resistance

Question 6

How can you use plasmids for gene cloning?

Answer 6

Cut the gene and plasmid using restriction enzymes, join DNA with ligase to form a recombinant DNA molecule, introduce into bacterium, and select for the bacteria that have the recombinant DNA molecule

Question 7

Why clone DNA?

Answer 7

To produce useful proteins such as insulin, for genetic screening, for gene therapy, to find out about genes .

Question 8

Why would you use the polymerase chain reaction?

Answer 8

Amplification of target DNA, to investigate variation, to investigation small deletions or insertions, to amplify specific DNA fragments.

Question 9

What are the stages of polymerase chain reaction?

Answer 9

Changing temperature cycles which denature, anneal and polymerase DNA.

Question 10

What is the enzyme used in polymerase chain reaction?

Answer 10

thermostable DNA polymerase

Question 11

What are the two types of protein gel electrophoresis?

Answer 11

isolectiric focusing and sds page.

Question 12

What does isolectric focusing do?

Answer 12

Seperates proteins on the basis of their isolectric point

Question 13

What does SDS page do?

Answer 13

separates proteins on the basis of size.

Question 14

What does two dimensional electrophoresis or 2D page do?

Answer 14

Separates proteins on the basis of both size and charge.

Question 15

What is proteonomics?

Answer 15

The analysis of all proteins expressed by a gene.

Question 16

What is the process of a ELISA?

Answer 16

In an antigen coated well, a specific antibody binds to a antigen, and an enzyme linked antibody binds to a specific antigen, and then washed, substrate is added and converted to a coloured product and the amount of colour formation is specific to the amount of added antibody.

Question 17

What is the process of western blotting?

Answer 17

Nitrocellulose replica of a gel eletropherogram, binding of a primary antibody and then binding of a enzyme linked secondary antibody, which forms a immunoblot.

Question 18

What are the two types of enzyme assays?

Answer 18

A continuous and a discontinuous assay.

Question 19

What is a restriction enzyme?

Answer 19

Recognise and cut specific DNA sequences at restriction sites

Question 20

What are the three blotting techniques and what are they used for?

Answer 20

Southern blotting, used to detect dna structure, northern blotting, using to detect rna structure and western blotting, which involves the detection of protein structures using antibodies.

Question 21

What is the overall process of southern blotting?

Answer 21

DNA electrophoresis separates the strands of DNA, it isa subsequently trasmfered to a membrane and there is hybridisation of a specific DNA probe.

Question 22

Why would you use southern blotting?

Answer 22

To investigate gene structure (such as large deletions/ duplications), to investigate gene expansions or triplet repeats, to investigate mutations, especially within allele specific proteins such as for sickle cell disease, to investigate variation and genetic relationships as it can detect very small amounts of DNA, and allows us to detect DNA from specific mixtures.

Question 23

What is the physical process of performing a southern blot?

Answer 23

Separate fragments by gel electrophoresis, fragments are transferred to a solid support such as a nitrogen cellulose filter, soaked in a alkaline solution to denature dsDNA fragments, transferred to membrane by capillary action or electrophoretic transfer, filter is placed is solution with labelled dna probe, which will bind wherever the complementary sequence is found, and the filter can be washed to remove unlabelled probe, and analysed using photographic film.

Question 24

Does the probe have to be 100% complementary?

Answer 24

No, an 80% complementarity probe will bind less tightly and there can be a partial overlap and the probe will still bind.

Question 25

What is the use of a ddNTP in the Sanger chain termination method?

Answer 25

There is a H at the 3' end instead of an OH, but is so similar in structure it can be incorporated into the DNA sequence as normal, but once it is bound it will stop further phosphodiester bonds from forming.

Question 26

How is the danger chain termination method performed?

Answer 26

4 separate tubes are used with a unique ddNTP, with a primer to initiate dna synthesis, after incubation they are laid out of a gel using Separate lines for each test tube, which will separate fragments on the basis of size and you can read off the bands formed to see the sequence. Or you can use fluorescent ddNTPs in the same tube, and are separated on the same capillary gel and then read using a lazer.

Question 27

What is reverse transcriptase PCR?

Answer 27

Uses a RNA instead of a DNA template, cDNA copy of a RNA template is made before PCR.

Question 28

What is a mircoarray used for?

Answer 28

Investigating 1000s of genes in situ, particularly for determining differential gene expression in conditions such as cancer, as well as chromosome deletions etc.

Question 29

What is involved in performing a microarray?

Answer 29

cDNA is gathered from normal and the patients cells using RT-PCR from RNA, and both sets of DNA are labelled with different fluorescent markers. They are mixed and allowed to hybridise, and most colours in the array should be a sort of brown colour but if green or red is overly present it could indicate different levels of gene expression or missing chromosomes.

Question 30

Why would you use fish?

Answer 30

Investigating chromosome behaviour, eg anaphase lag, investigating chromosome number and chromosome structure, investigating genes in situ.

Question 31

How would you perform FISH?

Answer 31

Probe DNA, label with fluorescent dye, denature and hybridise, fluorescent spots on chromosomes.

Question 32

What are the key features of DNA fingerprinting?

Answer 32

It considers one region only, and is used to show family relations and evidence for genetic relationships, minisatillite PCR is used to shoe small tandem repeats and copy number variation.

Question 33

What conditions can karyotyping be used to detect?

Answer 33

Trisomies, monosomies and chromosome translaocations.

Question 34

What does karotyping involve?

Answer 34

Arrest cells in the mitotic metaphase, put on mircoscope slide and stain chromosomes to have a look at the chromosome pairing.

Question 35

What techniques can you use for gene analysis at a chromosome level?

Answer 35

FISH and karyotype analysis.

Question 36

What techniques can you use to analyse DNA at a nucleotide level?

Answer 36

DNA sequencing.

Question 37

What techniques can you use to anaylse DNA at a gene level?

Answer 37

DNA fingerprinting, Southern Hybridisation, Northern Hybridisation, RT-PCR, Microarray, DNA fingerprinting.