molecular techniques

Question 1

3 processes which allow analysis of DNA

Answer 1

1) restriction enzymes
2) DNA gel electrophoresis
3) PCR

Question 2

describe how restriction enzymes can be used to analyse DNA

Answer 2

restriction endonuclease cut certain sites on the DNA (the way they cut the DNA results in an overhang). There are specific sites for each restriction endonuclease and these sequences can be found using online programs (because we know full human genome sequence)

Question 3

How does DNA electrophoresis work?

Answer 3

DNA is put into an agarose gel. Because it is negatively charged it will move towards the anode (+) meaning they will be spirited upon size basis. After this DNA strands are stained so they fluoresce when UV light is shined.

Question 4

what is DNA electrophoresis used for

Answer 4

to investigate DNA fragment size (see if DNA has deletion etc)
Look at mutations (sickle cell)
Used for DNA fingerprinting (relationships between people)
Used in DNA cloning

Question 5

describe the process of cloning DNA

Answer 5

(useful in making proteins such as insulin)

1) gene is isolated using restriction endonuclease or reverse transcriptase
2) gene is interred into plasmid vector
3) the plasmid (with the recombinant DNA) is inserted into DNA in bacteria or other stable host
4) bacteria produces the cloned gene and this is isolated and collected

Question 6

other uses of DNA cloning

Answer 6

gene therapy
genetic screening
to find out what genes do

Question 7

Describe the process of PCR

Answer 7

DNA amplification.

1) DNA is heated to 95 degrees and h bonds are broken
2) cool back down to room temp allowing primers to aneal to DNA strand
3) after the primers have annealed this allows DNA polymerase to bind and a double strand to be synthesised.

Question 8

which type of DNA polymerase is used in par and why

Answer 8

tap polymerase because it is thermostable (works at high temps is from bacteria found in hot springs)

Question 9

why use PCR?

Answer 9

DNA profiling
investigate bas mutations
investigate mutations
all require other process but also require a large small which PCR can provide

Question 10

3 ways to analyse proteins

Answer 10

1) protein electrophoresis
2) immunoassays
3) enzyme-assays

Question 11

describe the process of protein electrophoresis

Answer 11

proteins can be seperated on basis of size shape and charge. Proteins run through gel electrophoresis. Then able to compare the banding of proteins from 2 different cells and can see if there is a higher conc of a specific protein in one cell compared to another. (the darker the stain/band the more protein). This may allow diagnosis of certain diseases

Question 12

what is isoelectric focusing

Answer 12

proteins are separated on the basis of charge. Put in test tube where there is a gradient of pH’s. An electric field is applied and proteins will migrate until they reach a pH which matches their pI. Stop migrating and stained to identify.

Question 13

what is SDS PAGE

Answer 13

separation based on size
all proteins are denatured so they are the same shape. This is done using SDS which breaks down proteins to secondary structure. B-ME breaks disulphide bonds 1st.
Then all proteins have something applied which gives them the same charge meaning they all move toward the anode. Now carry out gel electrophoresis

Question 14

what is 2D PAGE

Answer 14

proteins permeated by isoelectric point
this is used in disease diagnosis because able to see up regulation of certain proteins because shows all the individual proteins

Question 15

what is an immunoassay

Answer 15

using antibodies to bind to specific sites to identify certain proteins.

Question 16

describe how immunoassays work and used in western blotting

Answer 16

antibodies bind to specific targets produced by B lymphocytes. Nitrocellulose gel is used. Antibody binds to specific protein wanting to be identified (dyed blue) and then an enzyme will bind to the antibody resulting in a single blot where the specific protein is.

Question 17

what are radioimmunoassays?

Answer 17

The same principle but using radioactively labelled antibodies

Question 18

what do enzyme assays do

Answer 18

measure enzyme activity

Question 19

why are enzyme assays useful

Answer 19

finding out how active an enzyme is can be used to diagnose a disease. May be a marker for this disease.

Question 20

examples of how enzyme assays are used

Answer 20

amylase in blood is an indicator for pancreatitis and troponin 1 is found in blood after MI

Question 21

describe how Sanger sequencing works

Answer 21

dideoxynucleotides are incorporated into DNA replication but once bound DNA replication is terminated. These nucleotides each contain a different stain depending on which base they are. These DNA strands are then operated by size using gel electrophoresis and able to oder the sizes and bases to find base sequence

Question 22

describe how southern hybridisation works

Answer 22

DNA probes with radioactive or Uv markers bind to their complimentary sequences. This is then transferred to a sheet using southern blotting. This allows base sequences to be identified. If a base is not present in a gene due to a mutation the probe will not bind

Question 23

describe how reverse transcriptase works

Answer 23

1) mRNA is isolated
2) primer binds to poly A tail and reverse transcriptase enzyme replicates the mRNA strand going backwards using DNA nucleotides to form cDNA
3) the RNA strand is degraded and use single strand of cDNA to make complimentary strand
4) This is then replicated using PCR

Question 24

describe how microarray technology works

Answer 24

Used to compare 2 cells (one affected and one non affected). remove mRNA samples and using reverse transcriptase form 2 different sets of cDNA with different stains. Combine these and allow them to hybridise (bind to their complimentary strands on the microchip. Wash the microchip and use a laser to look at the different stains on different genes. Allows you to see which genes are expressed in the affected and unaffected cell.

Question 25

describe how DNA fingerprinting works

Answer 25

Looks at exons. Uses microsatleites which have different repeat lengths in each person. These microsatelties are identified by restriction endonuclease which are able to cut it out. The longer they are the more repeats there are. can compare banding using gel electrophoresis. used to identify people or in a paternity test

Question 26

Describe how karyotyping works

Answer 26

Karyotyping is analysing the shape, size and number of chromosomes to identify diseases such as downs syndrome

Question 27

describe how FISH works

Answer 27

FISH stands for Fluorescent in situ hybridisation. The whole chromosome is coloured using probes and hybridisation of these probes. It can be used to show the mutations a tumour is causing e.g the translocation of the different parts of genes onto different chromosomes

Question 28

ethical issues with people having their entire genome sequenced

Answer 28

The benefits of sequencing the whole genome would be that we are able to predict which diseases some people are more susceptible to. However, who then owns your DNA, will it lead to discrimination e.g with insurance.

Question 29

DNA profiling?

Answer 29

DNA profiling is the newer and more modern version of DNA fingerprinting which uses the most variable repeats called tandems

Question 30

What is hybridisation?

Answer 30

The process of forming a double stranded nucleic acid from joining two complementary strands of DNA or RNA