MGD Session 7 Molecular Diagnosis 1+2

Question 1

DNA Sequencing What is the difference between ddNTP’s and dNTP’s?

Answer 1

ddNTP’s are a variation of dNTPs lacking a 3’ OH group, so polymerisation cannot occur

Question 2

DNA sequencing

What does the structure of ddNTP mean for the DNA strands it is added to?

Answer 2

Strand terminates

Question 3

DNA sequencing Why does the use of ddNTP produce lots of new fragments at different lengths?

Answer 3

Strands terminate at different points depending on where ddNTP added at each part.

Question 4

How many test tubes used in DNA sequencing and why

Answer 4

Four, each with of type of ddNTP in (ddATP, ddGTP, ddCTP, ddTTP)

Question 5

DNA sequencing How can you measure size of strands produced?

Answer 5

Gel electrophoresis

Question 6

Why do DNA strands need to be denatured in gel electrophoresis?

Answer 6

So they have charge

Question 7

Explain process of DNA electrophoresis.

Adequately.

Answer 7

Lots of fragments of different sizes produced. Can all be electrophoresised to separate according to size. Position each test tube fragment comes in indicates sequence, as each of four wells is different nucleic acid.

Question 8

What are restriction endonucleases?

Answer 8

Bacterial enzymes that recognise and cut specific DNA sequences known as restriction sites

Question 9

What word can be used to describe the structure of many restriction sites, and how long are they?

Answer 9

Palindromes, 4-8 bp

Question 10

How do bacteria protect their own DNA from restriction endonucleases?

Answer 10

Methylation

Question 11

What does the cutting of a DNA sequence by restriction endonuclease leave?

Answer 11

Sticky ends

Question 12

How can sticky ends be joined?

Answer 12

Via DNA ligase

Question 13

What four things can be found out by combining electrophoresis and Restriction endonucleases?

Answer 13

Size Huge mutant variety of clones

Mutations

DNA variation

Gene cloning

Question 14

Gene cloning

What is a plasmid?

Answer 14

Small circular ring of DNA

Question 15

What property of a plasmid makes it a good vector?

Answer 15

Can transfer from bacteria to bacteria

Question 16

Via what method can you add a desired gene into a plasmid?

Answer 16

Use of restriction endonucleases on both the target gene and the plasmid. Complementary sticky ends will be produced, which will combine under influence of DNA ligase.

Question 17

What is the process called by which a modified plasmid is introduced into a bacteria

Answer 17

Transformation

Question 18

Why is it useful for a plasmid to take up an antibiotic resistant gene as well as a desired gene?

Answer 18

Means those bacteria who didn’t take up plasmid will be killed by specified antibiotic.

Question 19

Give three reasons for cloning genes

Answer 19

Useful proteins, find out what genes do, screen for bad genes

Question 20

Name a process of DNA analysis which can be used to work out structure of DNA at nucleotide level

Answer 20

DNA sequencing

Question 21

Name three methods of assesing DNA at the gene level

Question 22

Name two ways of assesing DNA at chromosome level

Answer 22

Karyotyping

FISH

Question 23

Name three ways in which proteins can be analysed

Answer 23

Protein electrophoresis
Immunoassays
Enzyme assays

Question 24

What is Gel electrophoresis used for?

Answer 24

to separate different sized DNA fragments.

Question 25

Where are the solution of different fragments place in gel electrophoresis?

Answer 25

In a well at the negative annode end

Question 26

What charge does DNA have, and why?

Answer 26

Negative, as acidic and has lost hydrogens

Question 27

Which way does DNA move in GE (gel electrophoresis)

Answer 27

Towards the positive anode

Question 28

Which move faster in GE, large or small fragments?

Answer 28

Small

Question 29

How do we know the size of DNA/Protein during GE by the distance it goes?

Answer 29

By using a reference sample of fragments of known size

Question 30

Name four things required by Gel electrophoresis and why

Answer 30

Gel – A matrix that allows separation of DNA fragments
Buffer – Allows charge on DNA samples across the gel
Power Supply – Generates charge difference across the gel
Stain – To identify the presence of the separated DNA

Question 31

What does PCR do?

Answer 31

amplifies DNA segments by repeated copying of the target DNA

Question 32

What is the name of the DNA polymerase used in PCR, and why is it used?

Answer 32

TAQ DNA polymerase, used as it can survive high temps of 96* needed to break DNA double bonds

Question 33

What do the pair of primers in PCR do?

Answer 33

Uniquely define region of DNA to be copied

Question 34

Give three stages of PCR and associated temperatures

Answer 34

1. Denaturation at high temperature (94 – 960C)
2. Renaturation (annealing) at lower temperature (50 - 650C)
3. DNA synthesis at medium temperature (75 - 800C)

Question 35

Give three investigations that can be provided via PCR

Question 36

What is the difference between protein and DNA gel electrophoresis?

Answer 36

Proteins move towards either annode or cathode, so separated by charge

Question 37

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