I. DNA & RNA | 18. Methods for the investigation of genetic mutations and polymorphisms (RFLP, allele- specific PCR, DNA-sequencing and primer extension)

Question 1

I. Background
1. If different alleles would produce PCR-products with different lengths, what could we figure?

Answer 1

If different alleles would produce PCR-products with different lengths, we could figure out which allele (the diseased or healthy one) is present in a patient.

Question 2

I. Background
2. In length polymorphisms, what is the difference between alleles?

Answer 2

In length polymorphisms, the various alleles just differ in length.

Question 3

I. Background
3. In point mutations/SNPs, what is the difference between the lengths of alleles?

Answer 3

In point mutations/SNPs, the length of the various alleles is identical

Question 4

I. Background
4. In point mutations/SNPs, the length of the various alleles is identical
=> how do we convert different alleles into different sized PCR-products?

Answer 4

By using different methods (PCR- products have different size/composition/chemical properties etc.)

Question 5

II. Restriction fragment length polymorphism (RFLP)
1. What is Restriction fragment length polymorphism (RFLP)?

Answer 5

Restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences

Question 6

II. Restriction fragment length polymorphism (RFLP)
2. How does RFLP analysis work?

Answer 6

• In RFLP analysis, the DNA sample is broken into pieces and digested by restriction enzymes (restriction endonucleases), and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.
• This can be used in e.g. localization of genes for genetic disorders and determination of risk for disease.
• RFLP can also be used to determine the genotype of an individual

Question 7

II. Restriction fragment length polymorphism (RFLP)
4. What is Restriction fragment length polymorphism (RFLP) based on?

Answer 7

RFLP is basically based on the usage of restriction endonucleases (natural bacterial enzymes), which are enzymes that bind and cleave the DNA at specific recognition sites -> differentiate between 2 alleles

Question 8

II. Restriction fragment length polymorphism (RFLP)
5. What are the steps of Restriction fragment length polymorphism (RFLP) analysis?

Answer 8

1. The restriction endonuclease recognizes 4-8 bp long palindromic sequences in the DNA double helix and cleave both strands by hydrolysis of phosphodiester bonds
   - Sticky ends = different length cleavage
   - Blunt ends = same length cleavage
2. The sequence without the SNV will generate the recognition site -> fragment will be cut (T-allele
   in picture)
3. The sequence that contains a SNV will destroy the recognition site -> fragment will
   not be cut (C-allele)
   => After the recognition of which allele will be cut and not, it is easy to separate these two with the help of gel electrophoresis

Question 9

II. Restriction fragment length polymorphism (RFLP)
6. What is the difference between Sticky ends and blunt ends?

Answer 9

• Sticky ends = different length cleavage
• Blunt ends = same length cleavage

Question 10

II. Restriction fragment length polymorphism (RFLP)
7. What is the difference between the sequence containing a SNV and sequence not containing SNV

Answer 10

• The sequence without the SNV will generate the
  recognition site => fragment will be cut (T-allele
  in picture)
• The sequence that contains a SNV will destroy the recognition site => fragment will not be cut (C-allele)

Question 11

II. Restriction fragment length polymorphism (RFLP)
8. What are the 3 steps of RFLP analysis?
!!! Give 1 sentence for each step

Answer 11

1st step is to amplify the region
2nd step is the RFLP
3rd step = gel electrophoresis

Sample 1 = CC (homozygote)
Sample 2 = CT (heterozygote)
Sample 3 = TT (homozygote

Question 12

III. Allele-specific PCR
1. What is Allele-specific PCR?

Answer 12

Allele-specific PCR is a technique based on allele-specific primers, which can be used to analyze SNP effectively.

Question 13

III. Allele-specific PCR
2. How does Allele-specific PCR work?

Answer 13

Two separate reactions mixtures are used:
- Primers are created that are either (1) complementary to one allele or (2) the other allele
- The primer anneals exactly to the polymorphic locus, and if it is perfectly complementary => it creates a perfect starting point for DNA polymerase = synthesis of the strand will start
- If the primer is not perfectly complementary = DNA polymerase cannot start its synthesis = DNA polymerase STOPS

Question 14

III. Allele-specific PCR
3. How many primers are used in Allele-specific PCR?

Answer 14

There are 3 primers used in this type of PCR

Question 15

III. Allele-specific PCR
4. What is the difference between 3’-exonuclease and 5’-exonuclease activity?

Answer 15

• 3’-exonuclease activity = proofreading: responsible for the high accuracy of replication. The enzyme recognizes if the last nucleotide added was incorrect
• 5’-exonuclease activity helps us to remove the
  Okazaki fragment in front of the enzyme

Question 16

IV. DNA sequencing
1. What is DNA sequencing?

Answer 16

• DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule.
• It includes any method/technology that is used to determine the order of the 4 bases (A, G, C, T) in a strand of DNA

Question 17

IV. DNA sequencing
2. What are the steps of Sanger sequencing?

Answer 17

• A chain-termination method requiring a ssDNA
  template, DNA primer, DNA polymerase, normal deoxynucleotides (dNTPs) and a modified di-deoxynucleotide (ddNTPs)
• The ddNTP terminates the DNA strand elongation, because these nucleotides lack a 3’-OH group – required for the generation of the phosphodiester backbone => inhibits DNA polymerase from continuing elongation
• Comparing the length of the terminated ssDNA by ddNTP, to the original strand, allows us to find the position of the base

Question 18

IV. DNA sequencing
3. How should we set up for Sanger sequencing in order to determine the position of 4 bases?

Answer 18

4 reaction mixtures are set up, to determine the positions of the 4 bases. Added to these are the DNA template with the primer, 4 natural nucleotides (dATP, dCTP, dGTP, dTTP) and one unnatural ddNTP
- The primers are usually radioactively labelled, so we can see the fragments in the gel electrophoresis
- When a modified ddNTP is incorporated, it will cause the DNA polymerase to stop the elongation of DNA
- The different segments can now be separated in the gel electrophoresis -> smaller fragments will move faster than other fragments -> possible to determine the position of the 4 bases

Question 19

V. Primer extension
1. What is the role of Primer extension?

Answer 19

Primer extension is used to determine the start site of transcription

Question 20

V. Primer extension
2. What does primer extension require?

Answer 20

This technique requires a radiolabeled primer which is complementary to a region near the 3’end of the gene

Question 21

V. Primer extension
3. What is the role of Primer extension?

Answer 21

• The primer is allowed to anneal to the RNA, and reverse transcriptase is used to synthesize cDNA (complementary DNA) from the RNA until it reaches the 5’ end of the RNA
• By running the product on a polyacrylamide gel, it is possible to determine the transcriptional start site, as the length of the sequence on the gel represents the distance from the start site to the radiolabeled primer
  => Basically: Label primers with different fluorescence to see which one binds to the
  mutation point -> possible to see what base is there