Biology: Recombinant DNA Technology (control of gene expression)

Question 1

describe the process of making a protein using DNA gene transfer and cloning?

Answer 1

1) isolation of DNA fragments for desired gene
2) insertion of DNA fragment into vector.
3) transformation (DNA put into host cells).
4) identification of successful host cells using gene markers.
5) growth/cloning of population of host cells.

Question 2

how is reverse transcriptase used to make gene copies?

Answer 2

• mRNA coding for gene removed.
• mRNA acts as a template for on which a single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase.
• cDNA isolated by hydrolysis of mRNA.
• double stranded DNA is formed on the template of cDNA.
• results in a copy of the gene

Question 3

how are restriction endonucleases used to remove a desired gene?

Answer 3

• recognise a specific set of bases.

- they recognise this as a cut site, they cut the strand of DNA here.

Question 4

what’s the difference between blunt ends and sticky ends?

Answer 4

Blunt ends: straight cut site

sticky ends: staggered cut site, can bind to other sticky ends. 4 unpaired bases are opposites to eachother.

Question 5

how can genes be made using a gene machine?

Answer 5

• base sequence of protein determined.
• amino acid order determined.
• amino acid sequence used work out complementary mRNA codons.
• DNA sequence complementary to mRNA is then worked out.

STARTS WITH THE PROTEIN, ENDS WITH THE DNA.

PROCESS:

• the DNA sequence is fed into the computer, checked for biosecurity etc.
• computer designs oligonucleotides (short sections of single-stranded DNA)
• computer builds oligonucleotides by joining single nucleotides together.
• oligonucleotides joined together to make a gene.
• PCR used to replicate the gene.
• gene inserted into bacterial plasmid.
• bacteria is a vector as it can store, replicate and transfer the gene.
• new gene checked for mistakes
• bacterial cells carrying suitable genes are cloned.

Question 6

how are vectors used in ‘in vivo’ cloning.

Answer 6

• vector used to clone desired gene
• transforation - mixing vectors with host.
• only some vectors will take up the plasmids. some plasmids close before DNA is added. some DNA forms its own plasmid.

Question 7

what are the benefits of using PCR to copy DNA fragments?

Answer 7

• fully automated.
• fast
• non-invasive
• just need DNA

Question 8

what is a problem with using PCR?

Answer 8

can amplify contaminated DNA

Question 9

describe the process of PCR

Answer 9

• DNA fragments, polymerase and primers are mixed and put into thermocycler.
• high temp. causes hydrogen bonds to break, results in separate strands of DNA
• cooled so primers bind to complementary bases at the end of DNA fragments.
• primers provide the starting sequence for double-stranded DNA and prevent strands from rejoining.
• DNA polymerase makes double strands.
• temperature increased for DNA polymerase
• begins at the primer until it reaches the end of the fragment.

Question 10

what are DNA probes and what do they do?

Answer 10

• short, single stranded sections of DNA, bind to complementary sections of other DNA strands.
• marked to make bound sections identifiable.

Question 11

how can probes be used for genetic screening?

Answer 11

1) DNA fragments complementary to mutant allele are made.

2) DNA probe made by labelling fragment.
3) PCR used to copy probe
4) probe added to single-stranded fragment of patient DNA
5) if patient has a mutant allele, the probe will bind.
6) fragments labelled with probe, x ray used to detect them
7) sample washed to remove unbound probes.

Question 12

how can probes be used for cancer screening?

Answer 12

• can show oncogene mutations.
• can show single cancer cells
• can identify people at risk

Question 13

what is gene counselling? what are its uses?

Answer 13

• screen individuals with family history of a disease.
• probability of children with the disorder can be determined.
• allows individuals to make informed decisions.