9.1 Fragmentation DNA

Question 1

When was an accurate sequence of the Human genome completed?

Answer 1

2003

Question 2

By 2013 the genes how many species had been sequenced?

Answer 2

> 7000

Question 3

What are the 5 general ideas behind genome sequencing:

Answer 3

1. Fragmenting the genome
2. Cloning DNA fragments
3. Sequencing DNA fragments
4. Reconstructing the genome sequence from fragments
5. Analyzing the information found in genomes

Question 4

The job of each restriction enzyme?

Answer 4

To recognise a specific sequence of bases anywhere within the genome, it severs two phosphodiester bonds at the sequence, One in the sugar-phosphate backbone of each strand.

Question 5

The fragments generated by restriction enzymes are referred to as?

Answer 5

Restriction fragments

Question 6

The act of cutting DNA is called?

Answer 6

digestion

Question 7

So what is the function of restriction enzymes in bacteria where they naturally originate from?

Answer 7

They digest viral DNA to protect prokaryotic cells from viral infection.

Question 8

Bacteria shield their own genomes from digestion by these restriction enzymes through the selective addition of?

Answer 8

methyl groups (-CH3) to the restriction recognition sites in their genomic DNA

Question 9

A restriction enzyme is a _______________ isolated from bacteria

Answer 9

protein

Question 10

Recognition sites for restriction enzymes are usually _____________of double strand DNA

Answer 10

4-8 bp (Base pairs)

Question 11

Explain the two other properties of the recognition sites of restriction sites

Answer 11

1. Often palindromic- base sequences of each strand are identical when read in the 5’-3’ direction
2. Each restriction enzyme cuts at the same place relative to its specific recognition sequence

Question 12

LEARN THE 10 COMMONLY USED RESTRICTION ENZYMES

Answer 12

(Screenshoted)

Question 13

What are the two possible ways that the restriction enzyme will cut in and what kind of ends does it result in?

Answer 13

1. they cut straight through both DNA strands right at the line of symmetry to produce fragments with blunt ends
2. The cut is displaced equally in opposite directions from the line of symmetry by one or more bases to generate fragments with single-stranded ends also known as sticky ends. The ends have either 5’ overhangs or 3’ overhangs.

Question 14

Why does the second kind of cut produce fragments that are considered sticky?

Answer 14

because they are free to base pair with a complementary sequence from the DNA of any organism cut by the same restriction enzyme

Question 15

Are restriction enzymes endonucleases or exonucleases

Answer 15

they are endonucleases

Question 16

State what the use of restriction enzymes is

Answer 16

They are used to precisely cut DNA into smaller fragments, which is useful for DNA cloning, genetic mapping, and DNA analysis.

Question 17

Different restriction enzymes produce fragments of different ____________

Answer 17

lengths

Question 18

What is the formula for calculating the average fragment length (in bp)? Also, explain the formula for calculating the exact number of fragments

Answer 18

formula = 4^n
where “n” is the number of bases in the recognition site.
eg1: 4-base recognition site occurs every 4^4 bp. Therefore the average restriction fragment size is 256 bp. 3 billion bp genome/256bp= 12 million fragments
eg2: 6-base recognition site occurs every 4^6 bp= 4096bp. Meaning the average restriction fragment size is 4100bp (4.1 kb). 3 billion bp genome/ 4100 = 700 000 fragments

Question 19

It is important to note that the number of base pairs in a recognition site determines the average________

Answer 19

restriction fragment size

Question 20

_________________ can be used to fragment DNA at random locations rather than at the same location like restriction enzymes

Answer 20

Mechanical forces

Question 21

What are the two mechanical forces that cut DNA randomly?

Answer 21

1. Passing DNA through a thin needle at high pressure
2. Sonication (ultrasound energy)

Question 22

Gel electrophoresis separates DNA fragments according to?

Answer 22

size

Question 23

What does electrophoresis mean?

Answer 23

The movement of charged molecules in an electric filed

Question 24

Explain the whole process of Gel electrophoresis

Answer 24

1. Prepare an agarose gel for electrophoresis by pouring heated molten agarose into an acrylic plate to which a comb has been attached.
2. Allow the gel to cool and harden
3. Place the gel in a buffered aqueous solution.
4. Remove the comb and load DNA samples into well in the gel.
5. Apply the electric current
   (DNA has a negative charge so it moves toward the positive charge)
6. After electrophoresis, visualise DNA fragments by staining gel with fluorescent dye, and photograph gel under UV light
7. Determine the size of unknown fragments by comparison with migrating of DNA markers of known size (linear DNA fragments migration distance trhough the gel depends on size)