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Flashcards in MCBG 13+14 - Molecular techniques Deck (47):
1

How does hybridisation work? 3

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

2

Why was it named southern blotting?

Discovered by Prof Sir Southern

3

What is a Southern/Northern Blot. One word

Hybridisation. (Gel electrophoresis + hybrisation)

4

How is western blot different from southern blot?

Its uses proteins
DOES NOT USE HYBRIDISATION

5

Describe Western blot

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

6

Describe Southern Blot

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

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?

PCR (part of ) new-born’s HBB
gene

8

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

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

9

What is sickle-cell disease?
Whats the most common?

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

10

MstII is a restriction enzyme; where are restriction
enzymes found?

Bacteria

11

Whats a single-nucleotide polymorphism?

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

12

Name three diseases single-nucleotide polymorphism occur in?

CF
Sickle Cell Disease
Beta Thalassemia

13

What units make up haemoglobin

HbA = a2b2
Alpha and beta subunits

14

Sickle Cell Anaemia. What mutation occurs?

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

15

Sickle Cell Anaemia. Why does the mutation effect RBCs?

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

16

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

Glu6Val
or E6V

17

What type of genetic disease is it?

Autosomal recessive

18

Whats a mismatch mutation?

C=T

19

Whats a pyrimidine dimer?

MUTATION
Where two pyrimidines (C,U,T) bond

20

Whats an intercalating agent

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

21

Name sources of mutations
4/5

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

22

How is DNA read in transcription?

DNA is read 3’ to 5’

23

What direction is RNA synthesised in transcription?

mRNA synthesis 5’ to 3’

24

What direction is mRNA read translation?

mRNA is read 5’ to 3’

25

What direction is polypeptide chains synthesised?

polypeptide synthesis N to C
(REVERSE ALPHABETICAL)

26

Single nucleotide changes can be split into two types.

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

27

Whats a missense mutations?

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

28

Whats a non-sense mutations?

A mutation that causes a premature stop codon

29

Whats a silent mutation?

A mutation that doesnt effect the final mutation

30

Whats a conservative mutation?

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

31

Whats a non-conservative mutation?

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

32

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

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

33

Missense mutations come in two different types

conservative mutation
non-conservative mutation

34

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

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

35

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

Micro:
Insertion
Deletion
Substitution
Macro:
Duplication
Deletion
Translocation

36

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

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

37

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

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

38

How does DNA sequencing work? 4

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

39

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

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

40

What is reverse transcriptase PCR

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

41

How do microarrays work?

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

42

Fluorescent in situ hybridisation ??

Take DNA - Denature it
Add fluorescent probe
Allow hybridisation

43

What is the difference between an oncogene and a proto oncogene

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

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

Dominant would mean the baby would have the diseased phenotype

Normal would mean the baby had a normal phenotype

45

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

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

46

Give an example of a environmental factor that causes doublestrand breaks

Ionising radiation
X-rays
Anti-tumor drugs

47

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

UV light
Chemicals