Molecular Techniques

Question 1

What is a specific endonuclease?

Answer 1

Restriction enzymes produced by bacteria

Question 2

What do endonucleases do?

Answer 2

Recognise and degradation of foreign DNA

recognise and cut specific DNA

Question 3

What is special about an endonuclease?

Answer 3

Mainly palindromes

Question 4

What are restriction enzymes?

Answer 4

Molecular scissors

Question 5

What is DNA gel electrophoresis?

Answer 5

DNA is separated on the basis of size or shape

Question 6

What does DNA gel electrophoresis need to work?

Answer 6

Gel- a matrix that allows separation of DNA fragments
Buffer- allows charge on the DNA samples across the gel
Power supply- Generates charge difference across the gel
Stain/detection- To identify the presence of the separated DNA

Question 7

Why is restriction analysis used?

Answer 7

Investigate size of DNA, mutations, DNA variations, clone DNA

Question 8

What is a plasmid?

Answer 8

Small circular dsDNA
In bacteria
Can transfer to other bacteria
can have antibiotic resistance

Question 9

What are the basic steps of gene cloning?

Answer 9

Isolate gene with digestion enzymes
Insert gene into plasmid to form recombinant DNA
Introduce recombinant DNA molecule into suitable cells
identify and isolate the clone contain DNA of interest

Question 10

Why clone human genes?

Answer 10

To make useful proteins e.g. insulin

to find out what genes do, genetic screening, gene therapy

Question 11

How is proinsulin produced?

Answer 11

Proinsulin mRNA is reverse transcriptase into proinsulin cDNA
This then joins to a plasmid to make a recombinant plasmid
Then place into bacterium

Question 12

What are the steps of PCR

Answer 12

1. Heat to 95 degrees
2. Denature DNA by breaking hydrogen bonds
3. Heat to 60 degrees so primers can form hydrogen bonds and anneal with complementary sequences
4. Heat to 72 degrees so Taq polymerase adds nucleotides to the 3’ end
5. repeated so there will be two double stranded copies of the target DNA
6. Half of fragments will have flagging DNA as well as target DNA

Question 13

Why use PCR?

Answer 13

Amplify specific DNA fragments
investigate single base mutations
investigate small deletions or insertions
investigate genetic relationships

Question 14

what is protein gel electrophoresis?

Answer 14

Proteins can be separated based on size, shape or charge

Question 15

What is SDS-PAGE?

Answer 15

separation of proteins based on size

Question 16

What is IEF?

Answer 16

Seperation of proteins based on charge

Question 17

What is 2D-PAGE?

Answer 17

Allows separation of complex mixtures of protein

Question 18

How can you identify a protein?

Answer 18

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

Question 19

What is Proteomics?

Answer 19

analysis of all proteins expressed from genome

Question 20

What is molecular diagnosis?

Answer 20

analysis of a single purified protein

Question 21

Why are antibodies important?

Answer 21

Bind to specific proteins (antigens) and recognise a few amino acids on a protein (epitope)

Question 22

What is a polyclonal antibody?

Answer 22

produced by many B lymphocytes, multiple different antibodies, specific to 1 antigen, multiple epitopes

Question 23

What is a monoclonal antibody?

Answer 23

produced from 1 B lymphocyte, 1 identical antibody, specific to 1 antigen, 1 epitope

Question 24

What is a western blotting?

Answer 24

Uses antibodies to detect proteins

Question 25

What is an enzyme assay?

Answer 25

Add samples of enzyme to substrate and measure rate of reaction - can determine how active an enzyme is

Question 26

How can you detect mutations at a nucleotide level?

Answer 26

DNA sequencing
PCR
Restriction analysis

Question 27

How can you analyse DNA at a gene level?

Answer 27

Southern or Northern hybridisation
RT-PCR
Microarray
DNA fingerprinting and DNA profiling

Question 28

How can you analyse DNA at a chromosome level?

Answer 28

Karyotyping

FISH- chromosome painting

Question 29

How is DNA sequencing done?

Answer 29

Use tagged Dedioxy bases (have a H instead of an OH so chain cannot continue)
These bases are marked and electrophoresis is done to determine DNA order
Important for Genome sequencing https://www.youtube.com/watch?v=SRWvn1mUNMA

Question 30

What is allele specific PCR?

Answer 30

Use allele specific primers e.g. if we know a mutation in a gene causes a T, would use an A and if this would bind we know have the mutation

Question 31

What is southern hybridisation?

Answer 31

Used to determine if a specific nucleotide sequence is present
Done by using specific restriction enzymes
electrophoresis is done and transferred to a nylon filter
Radioactive nucleic acid probe is added
Probe binds to complementary DNA sequence

Question 32

What is northern hybridisation

Answer 32

Same as southern hybridisation but with mRNA

Less stable

Question 33

What is reverse transcriptase PCR?

Answer 33

Begin with mRNA rather than DNA

Question 34

What is microarray?

Answer 34

Use when you want to look at 1000s of genes at once
can have whole human genome on a chip
Used to compare two conditions- infected or uninfected

Question 35

What is DNA fingerprinting?

Answer 35

DNA is cut using a restriction enzyme
Fragments are then separated on the basis of size
Nylon filter is then used
DNA strands are denatured
Probes are added that are complementary to repeat sequences
Xray film
Where probes have bound- appear as dark bands
related- similar banding

Question 36

What is karyotyping?

Answer 36

Looks at the number and visual appearance of chromosomes
Chromosomes are paired up
can see banding patterns

Question 37

What is FISH?

Answer 37

Fluorescent in situ hybridisation

Chromosomes are painted so can easily see translocations and tumours