[21.1] producing DNA fragments

Question 1

why can organisms be genetically modified by a different species?

Answer 1

• genetic code is universal
• mechanisms of transcription and translation are essentially the same in all living organisms

Question 2

what is recombinant DNA technology?

Answer 2

artifically shifting a section of DNA from one genome to another

Question 3

what are the 5 stages in the process of making a protein using the DNA technology of gene transfer and cloning?

Answer 3

1. isolation of the DNA fragments that have the gene for a desired protein
2. insertion of the DNA fragment into a vector
3. transformation ie. transfer of DNA into suitable host cells
4. identification of host cells that have successfully taken up gene using gene markers
5. growth / cloning of population of host cells

Question 4

describe how can DNA fragments be produced? (3)

Answer 4

• using restriction endonucleases to cut fragments contianing the desired gene from DNA
• conversion of mRNA to cDNA using reverse transcriptase
• creating gene in gene machine, usually based on a known protein structure

Question 5

describe the process of producing DNA fragments using reverse transcriptase

Answer 5

1. isolate mRNA coding for gene you want
2. mRNA acts as a template where single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
3. cDNA is isolated by hydrolysis of mRNA with an enzyme
4. double-stranded complementary DNA (dscDNA) is formed on template of cDNA using DNA polymerase
5. copy of original gene is produced

Question 6

what do restriction endonucleases do?

Answer 6

hydrolyse phosphodiester bonds in the middle of a polynucleotide, cutting the sugar-phosphate backbone into sections at a specific sequence of bases called a recognition site

Question 7

what are the 2 different ways a DNA double strand can be cut?

Answer 7

• straight cut
• staggered cut

Question 8

what does a straight cut leave?

Answer 8

• blunt ends
• no unpaired bases

Question 9

what does a staggered cut leave?

Answer 9

• sticky ends
• unpaired bases which can lead to free-floating nucleotides
• palindromic sequences

Question 10

describe how genes are manufactured in a lab with a ‘gene machine’ (11)

Answer 10

1. desired nucleotide base sequence is determined from desired protein
2. AA sequence of this protein is determined
3. determine mRNA codons and work out complementary DNA triplets
4. feed desired sequence of nucleotiede bases for the gene into a computer
5. sequence is checked for biosafety and biosecurity to ensure it meets international standards and various ethical requirements
6. computer designs a sequence of small, overlapping single strands of nucleotides (oligonucleotides) which can be assembled into the desired gene
7. in an automated process, oligonucleotides are assembled by adding one nucleotide at a time in the required sequence
8. oligonucleotides are joined together to make a gene, which doesn’t have introns or other non-coding DNA
9. gene is replicated by PCR
10. using sticky ends, gene is inserted into bacterial plasmid, which acts as a vector for the gene to be stored, cloned or transferred to other organisms in the future
11. genes are checked using standard sequencing techniques. those with errors are rejected