recombinant DNA technology

Question 1

what is meant by recombinant DNA technology

Answer 1

the transfer of DNA fragments from one organism to another

Question 2

why does recombinant dna technology work?

Answer 2

because the genetic code is universal and therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms

Question 3

list the ways of isolating target genes

Answer 3

1. gene machines
2. reverse transcriptase
3. restriction enzymes

Question 4

outline the process of using reverse transcriptase to produce dna fragments

Answer 4

• mRNA complementary to the target gene is used as a template
• it is mixed with free nucleotides which match up to their base pairs and reverse transcriptase
• which forms the sugar phosphate backbone
• to create cDNA

Question 5

does cDNA contain introns

Answer 5

no

Question 6

outline the process of using restriction enzymes to produce dna fragments

Answer 6

• restriction endonucleases cut DNA at specific sequences (pallindronic sites / restriction sites)
• restriction sites on either target gene allow restriction enzymes to cut the target gene out
• leaving sticky ends

Question 7

what are sticky ends

Answer 7

unpaired bases

Question 8

outline the process of artificially synthesising a gene

Answer 8

• use a gene machine to make DNA from scratch
• 25 nucleotides joined at once to form oligonucleotides
• join oligonucleotides together to form a synthetic gene

Question 9

when isolating target genes what must be added?

Answer 9

• promoter region
• terminator region
• sticky ends
• a marker gene

Question 10

what is a plasmid

Answer 10

a double loop of DNA used to transfer genes between bacteria

Question 11

outline the process of inserting a DNA fragment into a vector

Answer 11

• a plasmid is used as the vector
• and is cut using the same restriction enzymes as the DNA so that the ends are complementary
• DNA ligase joins the fragment and plasmid together by reforming phosphodiester bonds
• forming recombinant DNA

Question 12

outline the process of inserting a vector into a host cell

Answer 12

• the host cells are mixed with the vectors in an ice-cold solution of calcium chloride
• then heat shocked to encourage the cells to take up the vectors by increasing the permeability of the bacterial cell wall
• the cells can be grown and the DNA fragment will be cloned

Question 13

what is a transgenic organism

Answer 13

organism containing recombinant DNA

Question 14

outline the process of identifying transformed cells using UV light

Answer 14

• marker genes coding for fluorescence can also be inserted into vectors along with the dna
• when cells begin to grow, UV light can be used to identify which cells have been taken up by the vector and which haven’t
• you can select and culture the transformed bacteria

Question 15

outline the process of identifying transformed cells using antibiotic resistance

Answer 15

• marker genes coding for antibiotic resistance can be inserted into vectors along with DNA
• when cells begin to grow, antibiotics can be used to identify which cells have taken up the vector (they won’t die, will be antibiotic resistant) and which haven’t (they will die)
• you can select and culture the transformed bacteria