Gene technology

Question 1

Stages in producing a protein using DNA technology

Answer 1

Isolation of DNA fragments that produce protein
Insertion of DNA into vector
Transfer of DNA into host cell
Identification of cells that successfully took up DNA using host markers
Growth and cloning

Question 2

How to use reverse transcriptase to convert mRNA to cDNA

Answer 2

Cell that produces protein is selected as they have large quantity of the mRNA. Reverse transcriptase converts mRNA to cDNA.
DNA polymerase builds up complementary nucleotides on cDNA to produce double stranded DNA

Question 3

How to use restriction endonucleases to produce DNA fragments

Answer 3

Cuts gene out leaving sticky ends

Question 4

How to use gene machine to produce DNA fragments

Answer 4

Amino acid sequence is determined and then mRNA codons are looked up and comp DNA triplets worked out.
Sequence of nucleotides fed into computer and assembled by oligonucleotides. Polymerase chain reaction constructs complementary strand and copies gene many times.
Inserted into plasmid using sticky ends

Question 5

Advantages of gene machine

Answer 5

Any sequence can be produced in short time.

Free of introns so can be transcribed by prokaryotes

Question 6

Preparation of DNA fragment and plasmid for insertion into vector

Answer 6

Promotor and terminator gene sequence added for attachment of RNA polymerase and transcription factors and for stopping of transcription.
Same restriction enzyme that cut out gene cuts open plasmid to create complementary sticky ends and joined using DNA ligase.

Question 7

Why do some cells not take up plasmid

Answer 7

Only 1% take up plasmid with desired gene.
Some plasmids close together again.
DNA fragments forms its own plasmid.

Question 8

How to find which bacterial cells taken up plasmids (but not necessarily gene)

Answer 8

Bacterial cells grown on medium containing ampicillin and therefore bacterial cells that taken up plasmid will have gene for resistance. These can break down ampicillin and survive and others will die.

Question 9

How to identify cells that taken up plasmid but no gene and eliminate them

Answer 9

Use of a marker gene (fluorescent, antibiotic resistance, enzyme action).

Question 10

How to use antibiotic resistance marker gene to identify if gene has been taken up

Answer 10

Incorporate gene into plasmid in the middle of antibiotic resistance gene so it stops producing enzyme. Use replica plate to identify to not kill original.

Question 11

How to use fluorescent marker gene to identify if gene has been taken up

Answer 11

Bacterial cell that taken up plasmid will not be able to produce GFP. Retain those that do not fluoresce

Question 12

How to use enzyme marker gene to identify if gene has been taken up

Answer 12

Gene that produces lactase that turns colourless substrate blue. Gene transplanted into lactase gene. Bacterial cells that taken up gene will not change colour of colourless substrate to blue when grown on it.

Question 13

How are plasmids inserted into bacteria

Answer 13

Calcium ions and heat shock make membrane permeable

Question 14

Primer

Answer 14

Short sequence of nucleotides that have set of bases complementary to those at one end of each of two DNA fragments

Question 15

Separation of DNA strand in PCR

Answer 15

DNA fragments, primers and DNA polymerase played in thermocycler and at 95C so hydrogen bonds break

Question 16

Annealing of primers in PCR

Answer 16

Mixture cooled to 55C so primers anneal to complementary bases at end of DNA. Provide starting point for DNA polymerase to begin copying and prevent strands re-joining.

Question 17

Synthesis of DNA in PCR

Answer 17

Temp increased to 72C and DNA polymerase adds complementary nucleotides along each separated DNA strand until reaches end of chain.

Question 18

How does PCR loop

Answer 18

Thermocycler increases temp to 95 at the end to break hydrogen bonds of copies to repeat again.

Question 19

Advantages of PCR

Answer 19

Extremely rapid (but will also clone other random DNA)
Does not require living cells so no culturing.

Question 20

Advantages of in vivo gene cloning

Answer 20

Vectors used so can easily introduce gene into organism.

No risk of contamination as only complementary sticky ends cut by same restriction enzymes will be taken up by plasmid

Question 21

DNA probe

Answer 21

Short, single stranded length of DNA that has a label that makes it identifiable with base sequences that are complementary to base sequence of target DNA.

Question 22

How DNA probe works overview

Answer 22

DNA probe has complementary base sequences to target DNA.
Extract DNA and add restriction enzymes.
Separate DNA into fragments using electrophoresis.
Separate two DNA strands to expose bases.
Complementary DNA probe hybridises with gene.
Check for marker.

Question 23

How DNA hybridisation works in DNA probing

Answer 23

Heating DNA until double strand separates and then when cooled, complementary sections (probe and DNA) anneal.

Question 24

Locating specific alleles of genes

Answer 24

Extract DNA and add restriction enzymes.
Separate DNA into fragments using electrophoresis.
Treat DNA (heating) to form single strands and expose bases.
Complementary DNA probe hybridises with gene.
Check for fluorescence or use X-ray film to find bound probe.

Question 25

Advantages of genetic screening using probing

Answer 25

Many different DNA probes can be fixed and so can test DNA simultaneously for many different disorders.
Mutated tumour suppressor genes can be identified so people can make informed decision also.
Personalised medicine.

Question 26

Genetic fingerprinting

Answer 26

Non-coding bases of DNA has VNTRs (variable number tandem repeats) which are different for each individual in number and length.

Question 27

Gel electrophoresis

Answer 27

Separates DNA fragments by size. Larger fragments, slower they move and so smaller fragments move further.

Question 28

Gel electrophoresis - digestion

Answer 28

DNA cut into fragments by restriction enzymes

Question 29

Gel electrophoresis - separation

Answer 29

DNA are separated by size according to gel electrophoresis under voltage and then gel immersed in alkali to separate strands.

Question 30

Gel electrophoresis - hybridisation

Answer 30

DNA probes which complementary base pair sequence to VNTRs bind with VNTRs.

Question 31

Gel electrophoresis - development

Answer 31

X-ray film exposed by radiation from proves and positions are located visually
Series of bars revealed