3.8.4 Gene technologies

Question 1

What is recombinant DNA technology?

Answer 1

• transfer of fragments of DNA from one organism to another
• possible because genetic code is universal

Question 2

Describe the reverse transcriptase method for isolating DNA fragments

Answer 2

• start with mRNA
• use reverse transciptase enzyme to create cDNA (complementary DNA), which is single stranded
• use DNA polymerase to create double stranded DNA
• ends in a gene with no introns, no sticky ends

Question 3

Describe the restriction endonuclease method for isolating DNA fragments

Answer 3

• starts with DNA
• cut out the gene using restriction endonucleases
• restriction endonucleases are enzymes that cut at specific recognition sites
• which are palindromes
• to leave blunt or sticky ends
• results in gene WITH introns

Question 4

Describe the gene machine method for isolating DNA fragments

Answer 4

• starts with polypeptide
• use gene machine to synthesise required DNA sequence
• can add promoter and terminator regions and restriction endonucleases sites on either side of DNA base sequence
• no enzymes required
• no introns, yes sticky ends

Question 5

What does PCR stand for?

Answer 5

Polymerase chain reaction

Question 6

What 4 things are added to the test tube before it enters the thermocycler (pcr) machine?

Answer 6

• DNA fragment to be copied
• DNA polymerase - to join adjacent DNA nucleotides in a phosphodiester bond to make complementary DNA strand
• primers - to anneal the DNA fragments and provide a starting attachment point for DNA polymerase
• DNA nucleotides - monomers to make the complementary strand of DNA to the original DNA fragment

Question 7

Describe the PCR process

Answer 7

• DNA heated to 95C
• to break the hydrogen bonds between complementary DNA base nucleotides by heat, so the stands separate
• cooled to 55C
• annealing (binding) of primers
• to provide a starting point for DNA polymerase to extend from
• nucleotides attached
• by complementary base pairing
• 72C
• optimum temperature for DNA polymerase
• DNA polymerase joins adjacent nucleotides together with phosphodiester bonds
• cycle repeats

Question 8

Compare the similarities of a probe and a primer

Answer 8

• both short
• single stranded DNA sequences
• complementary to DNA target sequence of interest

Question 9

What are the differences between a probe and a primer

Answer 9

• probe has a fluorescent or radioactive label, primer has no label
• probe used to identify presence of DNA fragment/gene, primer used to start DNA synthesis by acting as a binding site for DNA polymerase

Question 10

What are the facts genetic fingerprinting relies on?

Answer 10

• each individuals DNA is unique
• 95% of DNA is introns and non-coding sections between genes
• non coding DNA have hypervariable regions that contain repetitive sequences- VNTR sequences (mini/microsatellites)
• these vary in length and have a unique pattern

Question 11

Genetic fingerprinting process

Answer 11

• DNA extracted from sample, PCR amplifies DNA quantity
• DNA cut into sections using restriction endonucleases
• must leave required core VNTR sequences intact
• DNA fragments separated by gel electrophoresis according to length
• 2 strands of DNA separated by immersing gel in alkaline solution (dsDNA to ssDNA)
• southern blotting- transfer gel to nylon membrane, DNA fixed to nylon membrane with UV light
• radioactive probe added - only binds to complementary core VNTR sequences
• X Ray film/autoradiography to identify areas the probe has bound

Question 12

Uses of genetic fingerprinting

Answer 12

• forensic science
• paternity test
• determine genetic variability in a population
• medical diagnosis
• plant and animal breeding

Question 13

Gel electrophoresis process

Answer 13

• DNA negatively charged, moves towards positive electrode
• longer fragments move more slowly through gel, shorter fragments move more quickly
• distance moved depends on length/number of base pairs

Question 14

In vivo cloning: use of vectors

Answer 14

• restriction endonucleases cut at recognition sites leaving sticky ends
• DNA fragment modified to ensure transcription- promoter and terminator regions added
• insert DNA into a vector- plasmids:
• plasmid cut with same restriction endonuclease to create same sticky ends
• DNA fragment and plasmid combined with enzyme ligase (anneals them)
• plasmid with recombinant DNA inserted into host cell:
• Heat shock host cells to increase permeability of host cell membrane

Question 15

Using a radioactove probe

Answer 15

• extract DNA and add restriction endonucleases
• separate fragments using electrophoresis
• treat DNA to form single strands
• the probe will bind to the gene
• use autoradiography to show the bound probe