Recombinant DNA and Restriction Enzymes

Question 1

What is recombinant DNA (rDNA) technology?

Answer 1

• Techniques used to manipulate nucleic acids
• Manipulate DNA and RNA using: natural biochemical processes (e.g. enzymes), natural properties of microorganisms, biochemistry, microbiology

Question 2

What happens in rDNA technology?

Answer 2

• CUT and PASTE (splice) particular sequences
• CLONE sequences: make 10n identical copies (produce)
• ENGINEER sequences: study functions, create new functions, new products
• SEQUENCE DNA and RNA fragments
• LOCATE sequences /fragments using ‘probe’ hybridisation

Question 3

Units of RNA and DNA

Answer 3

• Nucleotides (nt): e.g. a gene 500 nt in length
• Bases (b): e.g. a sequencing read 400 bases long
• Base pairs (bp): e.g. a genome size 3.4 Gbp in size
• Average molecular weight: 330 Da/nt, 660 Da/bp

Question 4

Bacteria as the Workhorses of Molecular Biology

Answer 4

• Cheap and easy: manipulate, contain
• Exponential clonal growth: 1-2-4-8-16, etc, agar, suspension
• Yield limited by: available nutrients, available space
• Naturally move DNA
• Natural plasmid hosts and replicators
• Plasmids: vectors (carriers) of DNA: manipulation, production

Question 5

How does bacteria naturally move DNA?

Answer 5

• Conjugation: DNA transfer
• Transformation: DNA uptake
• Transduction: Phage transfer

Question 6

General approach to cloning

Answer 6

• cut vector, add DNA fragment, becomes rDNA molecule
• then enters bacterium
• transports into host cell
• rDNA molecule multiplies
• host cell divides
• numerous cell divisions resulting in clone

Question 7

Nucleic Acid Scissors: Nucleus

Answer 7

• Exonucleases: trim from the end (5’ or 3’, or both)

- Endonucleases: Cut internally

Question 8

Restriction Endonucleases

Answer 8

• Discovered by Luria (1950’s)
• Bacteriophages infect Strain A but not Strain B
• ”Restricted” to infecting strain A only

Question 9

Arber & Dussoix Model (1962)

Answer 9

• Certain bacteria contain enzymes that ‘digest’ foreign DNA: endonucleases
• Protect own DNA through chemical modification of DNA
• Methylation of bases A and C
• First enzymes isolated from E coli: EcoB and EcoK

Question 10

How do REs work/

Answer 10

• EcoK and EcoB: Cleave DNA remotely from their binding site, at random positions
• 1970: Haemohpilus influenzae (HindII): fixed recognition and cleavage site

Question 11

Type I RE

Answer 11

• Restriction & Methylation

- Cleaves remotely

Question 12

Type II RE

Answer 12

• Endonuclease OR Methylation

- Cleaves at/close to recognition site

Question 13

Type III RE

Answer 13

• Restriction & Methylation

- Cleaves close to recognition site

Question 14

RE Nomenclature

Answer 14

• HindIII
• H: genus
• in: species
• d: strain
• III: number (order in which it was discovered)

Question 15

RE Substrate: Recognition Sites

Answer 15

• REs recognise, bind and cut specific sites in dsDNA
• Sequence specificity per enzyme
• Recognition site: 4, 6 or 8 nt long (with exceptions)
• Often ‘palindromic’: In the case of restriction enzyme recognition sites: reads the same on both complementary strands of dsDNA when read in the 5’ to 3’ direction

Question 16

Ligases

Answer 16

• ‘Zipping’ together nucleic acids
• Enzymes that catalyse the ATP-driven ligation
• (ligate = to tie together) of nucleic acid backbones

Question 17

How Many RE cut sites (RES) per piece of DNA ?

Answer 17

• Depends on RES frequency in given DNA sample

- Estimation is possible based on: A, G, C, and T frequency, size of RES, length of DNA fragment/genome in question

Question 18

How Many RES per DNA fragment/genome?

Answer 18

• (𝑁_𝑡𝑜𝑡𝑎𝑙) can be calculated using: 𝑁_𝑡𝑜𝑡𝑎𝑙 = 𝑥^𝑦
• y = DNA sequence length of nucleotides
• 𝑥 = number of different possibilities for each nt position (A,G,C,T = 4)
• e.g. six consecutive nucleotides (𝑦=6) that are equally likely to be A, C, G or T: 𝑁_𝑡𝑜𝑡𝑎𝑙=𝑥^𝑦=4^6=4096

Question 19

Example calculation of REs fragment

Answer 19

• Each unique stretch of 𝑦 consecutive nucleotides occurs once every 𝑁_𝑡𝑜𝑡𝑎𝑙 bases
• e.g. EcoRI, for which 𝑦=6 (GAATTC), occurs once every 4096 bases
• The means that for a 100 kb genome, you expect to find on average: 100000/4096=24.4≈24 EcoRI sites (assuming each base is equally likely to occur)

Question 20

Restriction Digest Mapping

Answer 20

• Determine position of RES in a DNA sample
• Essential for cloning and genome analysis
• Use pure DNA sample
• Digest completely
• Size RES fragments by electrophoresis (E/P)

Question 21

Cut and Paste Use in Cloning: Somatostatin

Answer 21

• Hypothalamus and pancreas: reduce HGH, insulin, glucagon
• 14 aa’s: small and simple
• Known aa sequence
• 5 mg purification: 500,000 sheep brains
• E. coli production: unlimited (Itakura, 1977)

Question 22

Minimum requirements of DNA cloning

Answer 22

• Pure DNA
• Host organism
• DNA vector (plasmid, virus)
• Appropriate promoter, etc
• Restriction enzymes
• Ligase
• Recombinant host selection method

Question 23

Summary of steps in DNA cloning

Answer 23

• Obtain insert and vector
• Cut with appropriate RE
• Ligate into vector
• Transform into host
• Isolate recombinant(s)
• Purify DNA or TU product: RNA, Protein