OI

Question 1

How do you translate a strand that is given?

Answer 1

Usually the coding strand, unless stated otherwise, so use the codons left to right to find the corresponding amino acids
No need to reverse the strand or find the complement strand to uncode

Question 2

What does the normal COL1A1 sequence translate to?

Answer 2

Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly

Question 3

What is a common pattern

Answer 3

Glycine every 3rd

Question 4

What does the mutated version translate to?

Answer 4

Something

Question 5

Collagen structure?

Answer 5

Triple helix - like 3 tubes that wind around to make a rope, where the hydrogen (glycine’s ‘R’ group) slots in the middle

Question 6

Difference in molecular weights?

Answer 6

H - 1
Cysteine’s R group = 64
Very big difference

Question 7

Cysteine’s R group is much bigger, so how does that affect the winding of collagen?

Answer 7

Trying to wind up the 3 tubes which have cysteine’s ‘R’ group instead of glycine’s means the collagen isn’t wound as tightly
Bulge in the middle

Question 8

How do you separate molecules by electrophoresis in labs?

Answer 8

Either by charge OR size - generally, not both as that is complicated to compare

Question 9

How is the charge counteracted for the gel electrophoresis to test for collagen abnormalities?

Answer 9

Add detergent - SDS (

Normally protein exists in aqueous environment i.e. water, so the outside of the protein is hydrophillic

Question 10

How is the charge counteracted for the gel electrophoresis to test for collagen abnormalities?

Answer 10

Add detergent - SDS (sodium dodecyl sulphate) - 12 C chain with suphate on the end
Normally protein exists in aqueous environment i.e. water, so the outside of the protein is hydrophillic
Detergent is almost like an emulsifier, the carbon chain interacts with the hydrophobic part of the protein, turning it inside out, and coating the protein with the negative sulphate charge

Question 11

How does this help?

Answer 11

Charges on the proteins are all same and strongly negative

So essentially, the collagen is just being separated on size

Question 12

What is the difference in the gel electrophoresis between the use of 2-mercaptoethanol and not using 2-mercaptoethanol?

Answer 12

Can tell the difference vs cant tell the difference between the patient and control

Question 13

Why without 2-mercaptoethanol can you see the difference?

Answer 13

Cysteine only one to form disulphide bridges
In normal collagen, no disulphide bridges
In the mutated version, there are - 2x as big of a chain as 2 collagen structures are being held together by the disulphide bonds (the unsual gel electrophoresis band)

Question 14

Normally?

Answer 14

Can’t see the difference of just a 64 molecular weight as collagen is hundreds of thousands

Question 15

What does 2-mercaptoethanol do?

Answer 15

reducing

Question 16

So what does SDS do vs what does 2-mercaptoethanol do?

Answer 16

SDS - electrophoresis based purely on size - BUT does not break disulphide bonds
2-mercaptoethanol - reducing, breaks disuphide bonds
One with and one without, and they have different bands - some were held together by disulphide bonds

Question 17

What does the patient suffer from?

Answer 17

Long bone fractures

Question 18

What does collagen provide to bone strength?

Answer 18

The structure looks like overlapping small pieces of rods - provides structure
Uniform layer of calcium phosphate is deposited onto collagen

Question 19

Where is collagen found?

Answer 19

Cartilage and bones

Question 20

What makes bone different from cartilage?

Answer 20

Calcium

Question 21

How does the structure of the mutated collagen affect bone strength?

Answer 21

Collagen rods kinked - cannot pack as tightly when overlapping
Layer of calcium deposited is not as uniform - reduces the strength of the bone

Question 22

Collagen is made of which 3 rods?

Answer 22

Alpha 1 x2

Alpha 2

Question 23

What percentage of the collagen rods will be kinked? So why is this condition dominant?

Answer 23

Dominant - 2 Alpha 1s so random assortment can form the genotypes : MM, NM, MN, NN (M=mutated, N=normal)
75% produce the wrong structure assuming all
are equally transcibed, translated and enxorporated, 3/4 result in kinked rods

Question 24

How to test for the mutation in the foetus?

Answer 24

Sample from amniotic fluid
Small sample, therefore use PCR to replicate
Use to primers to section off the part of the DNA that contains the gene during PCR
PCR therefore replicates / amplifies just the segment of DNA required
Sequence the DNA / use gel electrophoresis -
Allow the DNA to anneal - pair up, if there is the mutation, there will be kinks - use electrophoresis to look for abnormal bands (different shaped bands)