21.1 Producing DNA fragments

Question 1

what is recombinant DNA

Answer 1

DNA of two different organisms that has been combined

Question 2

an organism that has had its DNA combine with another organism is known as

Answer 2

transgenic or genetically modified organism (GMO)

Question 3

5 stages of making a protein using DNA technology (gene transfer and cloning)

Answer 3

1. Isolation- of DNA fragments that have the gene for the desired protein.
2. Insertion- of DNA fragment into a vector
3. Transformation- transfer of DNA into suitable host cells
4. Identification- of host cells that have succesfully taken up the gene (by use of gene markers)
5. Growth/cloning- of the population of host cells

Question 4

3 methods of producing DNA fragments

Answer 4

1. Conversion of mRNA to cDNA using reverse transcriptase.
2. Using restriction endonucleases to cut fragments containing the desired gene from DNA
3. Creating the gene in a gene machine usually based on a known protein structure

Question 5

Using reverse transcriptase to produce DNA fragment

Answer 5

1. Cell that readily produces protein is selected
   (these cells have large quantities of relevant mRNA- easier to extract)
2. Reverse transcriptase makes DNA from RNA
   (known as complimentary/ cDNA. made up of nucleotides complimentary to RNA)
3. Enzyme DNA polymerase used to build up complimentary nucleotides on the cDNA template.
   (this double strand of DNA is the required gene)

Question 6

Using restriction endonucleases to produce DNA fragment

Answer 6

enzyme restriction endonuclease cuts up DNA

different types of restriction endonuclease- each one cuts a DNA double strand at a specific sequence of bases (recognition sequence)

sometimes between two opposite base pairs (leaves two straight edges- blunt ends)

sometimes cut in a staggered way. uneven cut. each strand has exposed unpaired bases (sticky ends)

Question 7

what is restriction endonuclease

Answer 7

an enzyme that cuts up DNA

Question 8

Using ‘gene machine’ to produce DNA fragment

1. How is the desired sequence of nucleotide bases of a gene determined

Answer 8

from desired protein we wish to produce

Question 9

advantages of ‘gene machine’

Answer 9

any sequence of nucleotides can be produced
short time (as little as ten days)
accurate
no introns or other noncoding DNA (can be transcribed and translated in prokaryotic cells)

Question 10

Using ‘gene machine’ to produce DNA fragment

2. What is done with desired sequence of nucleotide bases

Answer 10

fed into computer

Question 11

Using ‘gene machine’ to produce DNA fragment
3. What is the desired sequence of nucleotide bases checked for
(once it has been fed into the computer)

Answer 11

biosafety and biosecurity (meets international standards & ethical requirements)

Question 12

Using ‘gene machine’ to produce DNA fragment

4. What does the computer do

Answer 12

Designs small oligonucleotides which can be assembled into desired gene.

Question 13

Using ‘gene machine’ to produce DNA fragment

5. How are the oligonucleotides assembled

Answer 13

automated process- by adding one nucleotide at a time in the required sequence

Question 14

Using ‘gene machine’ to produce DNA fragment

6. How is gene replicated using polymerase chain reaction

Answer 14

polymerase chain reaction constructs complimentary strand of nucleotides to make required double stranded gene
then multiplies this gene many times to give numerous copies

Question 15

Using ‘gene machine’ to produce DNA fragment

7. Sticky ends are used when..

Answer 15

inserting the gene into a bacterial plasmid

Question 16

Using ‘gene machine’ to produce DNA fragment

8. Why is the gene inserted into a bacterial plasmid

Answer 16

acts as a vector for the gene allowing it to be stored, cloned or transferred to other organisms in the future

Question 17

Using ‘gene machine’ to produce DNA fragment

9. How are genes checked

Answer 17

using standard sequencing techniques. those with errors are rejected

Question 18

when the DNA is cut in a staggered way the exposed bases are

Answer 18

palindrome (two sequences are opposite to one another.
e.g one strand has TCGA
the other AGCT