Analysis Techniques

Question 1

With each blotting technique give name and what they use?

Answer 1

Southern- DNA
Northern- RNA
Western- proteins

Question 2

Describe process of DNA/Sanger Sequencing

Answer 2

Uses Fluorescently labelled dideoxy nucleotides. Once incorporated, they prevent elongation after them. After PCR you TF produce lots of DNA strands of different lengths. Can be read on gel electrophoresis

Question 3

What technique would you use to test for mutations present?

Answer 3

Restriction endonucleases, PCR, Southern blotting

Question 4

What is needed for and what would you use gel electrophoresis alongside?

Answer 4

Restriction endonucleases, agarose gel, salt buffer solution (to allow the charge to be carried), power supply (to generate charge difference across the gel)

Question 5

Why may you use restriction endonucleases?

Answer 5

To investigate size of DNA (check for deletions), check presence of mutations if enzyme binds/not, clone specific chunks of DNA

Question 6

What do restriction endonucleases recognise?

Answer 6

Specific palindrome sequences

Question 7

What can restriction endonucleases produce?

Answer 7

Sticky and blunt ends

Question 8

What is the function of the agarose gel?

Answer 8

Acts as a sieve, smaller fragments go further than larger ones.

Question 9

What enzyme is needed for PCR? Why?

Answer 9

Taq polymerase, it is able to withstand high temperatures

Question 10

What are the three main steps of PCR?

Answer 10

Heat to denature, cool with DNA primers to allow to anneal, reheat to allow the taq polymerase to work, repeat

Question 11

What is taq polymerase?

Answer 11

A thermos table DNA polymerase that can withstand high temperatures

Question 12

List some characteristics of probes

Answer 12

Don’t have to be a 100% match- can overlap.

Question 13

What is reverse transcriptase PCR? (RT-PCR)

Answer 13

As PCR doesn’t work with RNA, converts the RNA back to DNA

Question 14

What is microarray? And benefits

Answer 14

Can analyse several genes at once, give different DNA different colours showing either deletions (if less of one colour) etc

Question 15

What is DNA fingerprinting?

Answer 15

Where you compare the short random repeats of sequences that non-coding region of genes have (called VNTR- variable number tandem repeats). Only identical twins have same

Question 16

How may you asses chromosomal abnormalities?

Answer 16

Karotyping, FISH, chromosome painting,

Question 17

What is karotyping?

Answer 17

Visual picture of the full set of a persons chromosomes, organised according to chromosome number
The chromosomes are halted at metaphase to be able to visualise them

Question 18

What may you use karotyping for?

Answer 18

To spot aneuploidy, monoploidy, sex,

Question 19

What is FISH? Why is it so important?

Answer 19

Fluorescence in situ hybridisation, allows analysis to occur whilst chromosomes are still in the cell

Question 20

What different probes are used in FISH?

Answer 20

Telomeres, centromere, loci, whole chromosome

Question 21

Why may chromosomes painting be useful?

Answer 21

May be able to see where translocations may have happened

Question 22

What two types of prenatal testing are most commonly used?

Answer 22

Amniocentesis and chorionic villus sampling

Question 23

Which is more dangerous for miscarriage risk- amniocentesis or chorionic villus?

Answer 23

Chorionic villus sampling as its done earlier- weeks 11-12

Question 24

What happens in chorionic villus sampling?

Answer 24

Some chorionic villus cells are sampled by suction through a needle through the abdomen

Question 25

What happens in amniocentesis?

Answer 25

Sample of amniotic fluid taken by needle through abdomen, sample contains cells shed from baby and so can be used in karotyping

Question 26

Why can’t amniocentesis be done any earlier?

Answer 26

Not enough amniotic fluid and too many maternal cells

Question 27

Benefits of chorionic villus?

Answer 27

Results can be got quicker as cells are already dividing, can be done earlier TF if termination follows- safer for mother

Question 28

Why are metaphase chromosomes analysed?

Answer 28

Most condensed and easiest to visualise

Question 29

Down’s syndrome- defect and symptoms/signs

Answer 29

Trisomy 21, characteristic facial features, heart defects, increased incidence of leukaemia and Alzheimer’s, intellectual disability

Question 30

Defect and signs in Edwards

Answer 30

Trisomy 18, rocker bottom feet, overlapping fingers, small lower jaw, low set ears

Question 31

Defect and signs in Patau’s

Answer 31

Trisomy 13, polydactyly, cleft palate, die very young (few days)

Question 32

What is non-disjunction?

Answer 32

Occurs in meiosis division, forms a gamete with a missing or an extra chromosome

Question 33

What causes mosaicism?

Answer 33

Non-disjunction at a mitotic division

Question 34

What are two causes of aneuploidy?

Answer 34

Anaphase lag and non-disjunction

Question 35

What is anaphase lag?

Answer 35

In meiosis and mitosis, when chromosomes are pulled apart in anaphase, one chromosomes can be ‘left behind’ and may be lost

Question 36

What defect is turners and signs?

Answer 36

45 X, monosomie X, puffy feet, excess skin at back of neck ,short, heart defects, infertile

Question 37

What is a Robertsonian translocation?

Answer 37

When the Q arms of two acrocentric chromosomes fuse together to form one larger chromosome

Question 38

What happens in microarray?

Answer 38

Patient DNA compared to control DNA, both coloured differently, mix equal amounts of both and hybridise on a slide.. If they is no problem they will appear a mix, eg blue and red would make purple.

Question 39

How could you analyse a protein?

Answer 39

Protein gel electrophoresis, SDS-PAGE, Isoelectric focusing (separated on basis of charge/pI down pH gradient)

Question 40

What is SDS PAGE?

Answer 40

SDS is a detergent and so denatures protein and so the proteins can be separated purely on the basis of their Mr

Question 41

Enzyme assays use?

Answer 41

Measure activity of an enzyme eg lack or raised levels