Topic 8: processes

Question 1

using reverse transcription to produce fragments

Answer 1

• converts single strand mRNA into double stranded cDNA
• single strand cDNA isolated
• double strand DNA formed on cDNA template using DNA polymerase

Question 2

using restriction endonucleases to produce fragments

Answer 2

• binds to recognition site at palindromic sequence
• produces straight cut and blunt ends or staggered cut with sticky ends

Question 3

using the gene machine to produce fragments

Answer 3

• base sequence determined from desired protein amino acid sequence and put into computer
• sequence checked for biosafety
• oligonucleotides assembled into and joined to produce desired gene
• gene replicated using PCR and screened

Question 4

in vivo gene cloning

Answer 4

• restriction endonuclease cuts sticky ends on vector - complementary to fragment sticky ends so bind due to DNA ligase
• vector transfers recomb. DNA to host vis heat shock or injection
• GM cells identified with marker genes for antibiotic resistance, placed on agar so only those w recomb. DNA survive
• add promoter and terminator region

Question 5

in vitro: polymerase chain reaction

Answer 5

1. fragments, primers and DNA polymerase added to thermocouples and heated to 95°C to cleave H+ bonds
2. mixture cooled to 55°C so primers can anneal (allows DNA polymerase to bind and prevent DNA strands from rejoining)
3. mixture reheated to 72°C as optimum temp for DNA polymerase

Question 6

advantages of in vitro cloning

Answer 6

• very rapid and produces DNA on a large scale
• doesn’t require living cells

Question 7

advantages of in vivo

Answer 7

• useful to introduce a gene into another organism
• involves almost no risk of contamination
• it’s very accurate
• cuts out specific genes so precise
• produces transformed bacteria to produce large quantities of gene products

Question 8

locating specific alleles using DNA probes

Answer 8

1. determine sequence of mutant gene
2. fragment of DNA complementary to mutant allele produced
3. multiple copies formed using pcr
4. probes marked w/ fluorescent or radioactive tag
5. suspected DNA heated to separate strands then cooled and probes added
6. if mutant allele present probe will hybridise
7. DNA washed clean of any unattached probes
8. tag detected using UV light or x-ray

Question 9

producing a genetic fingerprint

Answer 9

1. DNA extracted from sample
2. restriction endonucleases cut DNA into fragments
3. fragments separated using gel electrophoresis and transferred to nylon membrane
4. probes with radioactive tag added to hybridise and label fragments
5. x-ray reveals bands

Question 10

gel electrophoresis

Answer 10

• DNA (up to 500 bases long) placed on agar at end with negative electrode, voltage applied across it
• resistance of gel means larger fragments move slower and less far towards positive electrode so different length fragments are separated