Ch 21

Question 1

Name a limitation of using restriction nucleases?

Answer 1

Product still contains introns

Question 2

Name a limitation of using a gene machine?

Answer 2

Need to know the amino acid sequence first

Question 3

Name a limitation of using reverse transcriptase?

Answer 3

More time consuming and difficult steps

Question 4

Describe what Recombinant DNA tech means

Answer 4

transfer of fragments of DNA from one organism to another

Question 5

Name the 5 stages of producing clones of cells with a desired gene

Answer 5

1) Isolation of the DNA fragment
2) Insertion of DNA fragment into a vector
3) transformation of DNA into suitable host cells
4) identification of the host cells that have taken up the gene via gene markers
5) Growth/cloning of population of host cell

Question 6

How can fragments of DNA be produced?

Answer 6

Conversion of RNA to DNA via reverse transcriptase
Use of restriction endonucleases of cut fragments containing desired genes
Use of gene machine

Question 7

Describe how reverse transcriptase is used to make fragments of DNA

Answer 7

mRNA is isolated containing the desired gene and acts as a template from the reverse transcriptase to produce cDNA
DNA polymerase is used ti create double stranded DNA

Question 8

Describe how restriction endonucleases are used to make fragments of DNA

Answer 8

Restriction endonucleases cut up viral DNA in bacteria at the recognition sequence to produce either blunt or sticky ends

Question 9

Describe how the Gene machine works

Answer 9

AA sequence is determined as are mRNA codons and DNA triplets
Info is put on computer and checked for biosafety and biosecurity
Computer produces oligonucleotides and assembles these to produce a gene with no introns

Question 10

What are oligonucleotides ?

Answer 10

small overlapping single strands of a nucleotide produced by a gene machine

Question 11

What reaction is used to insert DNA into a vector

Answer 11

Polymer Chain reaction

Question 12

What does ‘in vivo’ mean?

Answer 12

transfer fragments of a host cell via vector

Question 13

What does ‘in vitro’ mean?

Answer 13

polymerase chain reaction

Question 14

How do you prepare DNA fragment for insertion in in vivo gene cloning ?

Answer 14

Addition of extra lengths of DNA
Promoter = binding site of RNA polymerase
Terminator = site where RNA polymerase is released and trancitrpn ends

Question 15

What are recognition sites?

Answer 15

sequences of DNA that are cut by restriction nucleases

Question 16

Why use the same restriction endonucleases to cut DNA?

Answer 16

so fragment ends are complementary so ends are sticky and can later be joined by DNA ligase

Question 17

What is the role of DNA ligase?

Answer 17

Bind sugar phosphate backbone together

Question 18

How do you insert DNA fragment into a vector in in vivo gene cloning ?

Answer 18

Vector usually= plasmid

Endonucleases breaks the loop at an antibiotic resistant gene and DNA ligase incorporates them together

Question 19

How do you introduce DNA into a host cell in in vivo gene cloning ?

Answer 19

plasmid and bacterial cell are put in medium with Ca2+

Ions and increased temp makes bacterial membrane more permeable so plasmid can move into eh bacterial cytoplasm

Question 20

What is transformation?

Answer 20

introduce DNA into a host cell

Question 21

Name three ways introducing DNA into a host cell can fail

Answer 21

only small amounts take up the plasmids
some plasmids don’t take up the DNA fragments
Some DNA fragments ends join to form its own plasmids

Question 22

Name the three type of marker genes

Answer 22

Using antibiotic resistance
use of Fluorescent protein
Enzyme action that can be identified

Question 23

Describe how replica plating works

Answer 23

Add gene to antibiotic resistant gene so those who die in antibiotic have taken up the gene. Also can uptake another resistance so can tell if bacteria taken up plasmid

Question 24

Describe how GFP is used as a marker gene

Answer 24

no florescent = taken up gene

Question 25

Describe how lactaase is used as a marker gene

Answer 25

Lactase turns colourless substrate blue.

If DNA fragment inserted in this gene then remain colourless shows it has been taken in