MCBG 13+14 - Molecular techniques

Question 1

How does hybridisation work? 3

Answer 1

1. ) Denature ds DNA
2. ) Add a ssDNA or RNA probe (sequence of Nucleotides)
3. ) Eithe probe is fluorescent/radioactive or you add a label

Question 2

Why was it named southern blotting?

Answer 2

Discovered by Prof Sir Southern

Question 3

What is a Southern/Northern Blot. One word

Answer 3

Hybridisation. (Gel electrophoresis + hybrisation)

Question 4

How is western blot different from southern blot?

Answer 4

Its uses proteins

DOES NOT USE HYBRIDISATION

Question 5

Describe Western blot

Answer 5

1. Gel Electrophoresis
2. Blotting onto surface
3. Adding labelling probs (antibodies) No hybrisation

Question 6

Describe Southern Blot

Answer 6

1. Restriction enzymes cleave DNA
2. Gel Electrophoresis
3. Transfer to nylon
4. Hybridise with gene probe ( fluorescence or radioactive)
5. Detect labels

Question 7

Sickle cell disease is a mutation in the HBB
gene. What is the most suitable way to get the
new-born’s HBB gene for a genetic test?

Answer 7

PCR (part of ) new-born’s HBB

gene

Question 8

After you PCR what can you then do after? 2/3

Answer 8

1. restriction analysis
2. gel electrophoresis (Southern blotting)
3. DNA sequencing

Question 9

What is sickle-cell disease?

Whats the most common?

Answer 9

Group of blood disorders with the most common being sickle cell annemia.

Question 10

MstII is a restriction enzyme; where are restriction

enzymes found?

Answer 10

Bacteria

Question 11

Whats a single-nucleotide polymorphism?

Answer 11

A different base. A specific base position in the human genome, the base C may appear in most individuals, but in a minority of individuals it doesnt

Question 12

Name three diseases single-nucleotide polymorphism occur in?

Answer 12

CF
Sickle Cell Disease
Beta Thalassemia

Question 13

What units make up haemoglobin

Answer 13

HbA = a2b2

Alpha and beta subunits

Question 14

Sickle Cell Anaemia. What mutation occurs?

Answer 14

Single Base mutation(exchange).
A->T in the B-globin gene
Glutamate -> Valine

Question 15

Sickle Cell Anaemia. Why does the mutation effect RBCs?

Answer 15

Valine is hydrophobic. This distorts the shape of the cell. Causes it to collapse and form a sicle shape

Question 16

Sickle Cell Anaemia. What is the name of the of Mutation (found on a biomed form) [hint aa]

Answer 16

Glu6Val

or E6V

Question 17

What type of genetic disease is it?

Answer 17

Autosomal recessive

Question 18

Whats a mismatch mutation?

Answer 18

C=T

Question 19

Whats a pyrimidine dimer?

Answer 19

MUTATION

Where two pyrimidines (C,U,T) bond

Question 20

Whats an intercalating agent

Answer 20

hidium bromide
proflavine
molecules that may insert between bases in DNA, causing frameshift mutation during replication

Question 21

Name sources of mutations

4/5

Answer 21

1. Ionising radiation
2. UV light
3. Oxidising chemicals
4. inflammation
5. Smoking

Question 22

How is DNA read in transcription?

Answer 22

DNA is read 3’ to 5’

Question 23

What direction is RNA synthesised in transcription?

Answer 23

mRNA synthesis 5’ to 3’

Question 24

What direction is mRNA read translation?

Answer 24

mRNA is read 5’ to 3’

Question 25

What direction is polypeptide chains synthesised?

Answer 25

polypeptide synthesis N to C | REVERSE ALPHABETICAL

Question 26

Single nucleotide changes can be split into two types.

Answer 26

Transition - Purine to purine | Transversion - (A/G-C/T)

Question 27

Whats a missense mutations?

Answer 27

A mutation that causes any amino acid to be changed from its original

Question 28

Whats a non-sense mutations?

Answer 28

A mutation that causes a premature stop codon

Question 29

Whats a silent mutation?

Answer 29

A mutation that doesnt effect the final mutation

Question 30

Whats a conservative mutation?

Answer 30

A mutation that changes an amino acid to one that has similar properties

Question 31

Whats a non-conservative mutation?

Answer 31

A mutation that changes an amino acid to one that has different properties

Question 32

What type of mutation is sickle cell anaemia? Sense missense or non-sense

Answer 32

Non-conservative MISSENSE Mutation | (a point mutation that doesnt cause a FS but changes the amino acid GLU-> VAL. So its a MISSENSE Mutation

Question 33

Missense mutations come in two different types

Answer 33

conservative mutation | non-conservative mutation

Question 34

IMPORTANT. | What are the three (that i care about) Classification of mutations?

Answer 34

1. ) Effect on structure 2. ) Effect on protein sequence 3. ) Nomenclature

Question 35

In the classification that explains an effect on structure. What types of mutations are there? 6 (3+3)

Answer 35

``` Micro: Insertion Deletion Substitution Macro: Duplication Deletion Translocation ```

Question 36

In the classification that explains an effect on protein sequence. What types of mutations are there? 4

Answer 36

Frame Shift Missense (Conserve and Non-Conserve) Nonsense Silent

Question 37

In the classification that explains nomenclature. What types of mutations are there? 3

Answer 37

``` Nucleotide Substitution (76A>T) Amino acid Substitution (E6V) Amino acid deletion (/\F508) ```

Question 38

How does DNA sequencing work? 4

Answer 38

1. )Take DNA and Amplify the sample sequence using PCR (then denature it) 2. )Add Bases, DNA polymerase and fluorescently labelled di-deoxyribose nucleotides 3. ) Gel electrophoresis 4. ) Analyse using a computer and laser

Question 39

How does DNA finger printing work (in comparision to sequencing). Basically what are the steps? 3

Answer 39

It takes VNTRs (variable number tendem repeats) STR (short tandem repeats) amplifies them and then gel electrophoresis.

Question 40

What is reverse transcriptase PCR

Answer 40

Type of PCR | Uses mRNA instead of DNA and Reverse transcriptase enzymeto convert it into cDNA

Question 41

How do microarrays work?

Answer 41

Extract either DNA or mRNA from a cell (you tend to go for one normal and one diseased cell). If mRNA you need to reverse transcriptase PCR it and ad a lable. The mix of labelled GM is added to the microarray where HYBRIDISATION occurs

Question 42

Fluorescent in situ hybridisation ??

Answer 42

Take DNA - Denature it Add fluorescent probe Allow hybridisation

Question 43

What is the difference between an oncogene and a proto oncogene

Answer 43

A proto-oncogene is a normal gene that has many different functions in the cell, if it becomes mutated it will become a oncogene. Think of it like a pre-cursor Oncogenes are genes which can transform a cell into a tumour cell

Question 44

An extensive genetic test carried out on an unborn baby includes the DNA sequencing on an entire chromosome. The test reveals a frameshift mutation which leads to a premature stop codon on one of the alleles of a gene of known function. Explain in general terms what you expect to happen if the mutant allele is recessive to the wild type allele, and what if the mutant allele was dominant over the wild type allele

Answer 44

Dominant would mean the baby would have the diseased phenotype Normal would mean the baby had a normal phenotype

Question 45

Give an example of a environmental factor that causes single strand breaks

Answer 45

Ionising radiation X-rays Anti-tumor drugs Reactive oxidisation species

Question 46

Give an example of a environmental factor that causes doublestrand breaks

Answer 46

Ionising radiation X-rays Anti-tumor drugs

Question 47

Give an example of a environmental factor that causes pyrimidine dimers?

Answer 47

UV light | Chemicals