Recombinant DNA tech

Question 1

Recombinant DNA tech

Answer 1

• transfer of DNA fragments from one species/organism to another

Question 2

Why can transferred DNA fragments be used ?

Answer 2

Transferred DNA fragments can be transcribed and translated within the cells of the transgenic/recipient organism as the genetic code is universal and transcription and translation mechanism are also universal

Question 3

Recombinant DNA

Answer 3

• altered DNA with introduced nucleotides (usually from another organism)

Question 4

GMO

Answer 4

Any organism with introduced genetic material (recombinant DNA)

Question 5

Steps of genetic engineering

Answer 5

1. identification of desired gene/DNA fragment
2. Isolation of the desired gene/DNA fragment ( using either restriction endonuclease, reverse transcription, gene machine)
3. Amplification of the DNA fragment using PCR
4. Transfer of the DNA fragment into the organism using a vector
5. Identifying cells which have taken up the desired gene/DNA fragment by using marker genes

Question 6

Methods of producing DNA fragments

Answer 6

• restriction endonucleause
• reverse transcription
• gene machine

Question 7

Restriction endonuclease

Answer 7

• Different restriction endonuclease cut DNA at specific base sequences called recognition sites by hydrolysing phosphodiester bonds
• the shape of the recognition site is complementary to the active site of the restriction endonuclease
• recognition sites are palindromic (read the same in both directions)
  In this method, introns are not removed

Question 8

Sticky ends

Answer 8

• rescteiction endonuclease cut DNA in a staggered fashion forming sticky ends, where an uneven cut is left, in which each strand of DNA has exposed, unpaired bases

Question 9

Reverse transcription

Answer 9

1. mRNA is isolated from a cell that readily synthesis the protein coded for by the desired gene/DNA fragment
2. mRNA is mixed with DNA nucleotides and reverse transcriptase => reverse transcriptase uses mRNA as a template to synthesise a single strand of cDNA which does not contain introns
3. DNA polymerase forms a second strand of DNA using cDNA as a template and this produces a complete DNA fragment

Question 10

Reverse transcription advantages

Answer 10

• there is more mRNA in cells than DNA making mRNA more easily extracted
• in mRNA introns have been removed by splicing so can be translated/transcribed by a prokaryote who can’t remove introns by splicing

Question 11

Gene machine

Answer 11

• synthesises DNA fragments from scratch without the need for a pre-existing DNA template
• amino acid sequence of the protein of interest in determined allowing the mRNA base sequence to be established
• using this mRNA sequence, the complementary DNA sequence can be identified
• the gene machine then assembles short strands of DNA, one nucleotide at a time forming one complete DNA fragment

Question 12

Gene machine advantages

Answer 12

• DNA fragments are produced quickly and accurately
• DNA produced contains no introns so can be transcribed+ translated in prokaryotes

Question 13

PCR

Answer 13

• amplifies specific DNA fragments producing many copies (in vitro method)

Question 14

PCR reaction mixture

Answer 14

• heat stable DNA polymerase/TAQ polymerase
• DNA nucleotides
• complementary reverse and forward primers
• desired DNA fragment ( which acts as a template)

Question 15

PCR - step 1 : strand separation (95 degrees)

Answer 15

• heat to 95 degrees
• this separates DNA strands by breaking the hydrogen bonds between bases

Question 16

PCR - step 2 : Annealing/primer binding

Answer 16

• cool to 55 degrees
• this allows primers to bind to the DNA fragment template strands by forming hydrogen bonds between complementary bases

Question 17

PCR - step 3 : synthesis of DNA (72 degrees)

Answer 17

• heat to 72 degrees as it is the optimum temperature for DNA polymerase
• the free DNA nucleotides complementary base pair with the exposed bases on the template strands
• DNA polymerase joins adjacent nucleotides forming phosphodiester bonds in condensation reactions thus synthesising the new complementary DNA Strand

Question 18

Role of primers

Answer 18

• A primer is a short single stranded DNA fragment
• it is complementary to the template DNA at the start of the desired gene which allows DNA polymerase to bind to start synthesis
• this is because DNA polymerase can only add nucleotides onto pre-existing 3’ end
• thus two different primers are required (forward + reverse) because DNA strands run anti parallel but DNA polymerase can only run in one direction