Chapter 21 - Recombinant DNA technology

A-Level, AQA

what are the three ways to create DNA fragments:

1. gene machine

2. reverse transcriptase

3. restriction endonuclease

how does the gene machine create DNA fragments:

1. DNA sequence entered

2. biosafety and biosecurity checked

3. oligonucleotides formed

advantages of gene machine

1. quick

2. easy

how is reverse transcriptase used to make DNA fragments:

1. a cell that naturally produces protein of interest selected

2. these cells should have large amount of mRNA

3. reverse transcriptase joins DNA nucleotide to mRNA via CBP

4. single-stranded DNA made (cDNA)

5. to make it double-stranded DNA polymerase is used

advantages of using reverse transcriptase:

has no introns

how are restriction endonuclease used to create DNA fragment

1. cut desired gene with restriction endonuclease

2. forms sticky ends

what is the importance of sticky ends?

1. joins DNA together

2. forms complementary base pairs

describe how a gene of one organism could be removed and inserted into the cells of another organism

1. cut DNA at specific base sequence using restriction endonuclease

2. cut the plasmid open using same restriction endonuclease

3. sticky ends formed which join with desired gene via CBP

4. fix by DNA ligase

5. transfer plasmid to cells via heat shock and Calcium ions

definition of a vector

carries DNA into host cell

state the 3 types of marker genes:

1. Antibiotic resistance genes

2. fluorescent genes

3. genes for enzymes

what is the purpose of marker genes:

1. inserted into plasmid

2. to see which cells have taken up plasmid

explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected

1. isolate wanter DNA from another organism using restriction endonuclease

2. produces sticky ends

3. use ligase to join gene to plasmid

4. include a marker gene with antibiotic resistance

5. add plasmid to bacteria to grow

6. marker gene is expressed

7. bacteria not killed have antibiotic resistance and have taken up plasmid

what is the purpose of PCR

to amplify the DNA

describe the process of PCR

1. heat DNA to 95C to break hydrogen bonds

2. add primers

3. add nucleotides

4. cool to 55C to allow hydrogen bonds to reform and binding of nucleotides

5. heat to 72C and let DNA polymerase form phosphodiester bonds between adjacent nucleotides

6. repeat cycle many times

the purpose of primers in PCR:

1. to show region of DNA that needs to be copied

2. enzyme needs starting strand to attach nucleotides

what are the components needed for PCR

1. DNA fragment

2. primers

3. DNA polymerase

4. free nucleotides

5. thermocycler

key benefits of GMC

1. the process involves less labour

2. the process is cheaper

3. herbicide-resistant GM crops allow the use of herbicides to kill weeds without harming the crops

4. insect-resistant GM crops are less likely to be killed by pests

5. resistant to pests and herbicides increases crop yield

main advantages of genetic engineering in farm animals

1. improves quality

2. improves quantity

3. improves productivity

state the definition of DNA probes:

single-stranded DNA complementary to specific base sequence

how does DNA hybridisation work?

1. denaturation - DNA strands are heated causing them to separate into 2 single strands

2. DNA strands digested into fragments using restriction endonucleases

3. DNA fragments are mixed with DNA probes that are marked

4. DNA probes bind to complementary DNA strands

5. excess DNA probes removed by washing

6. UV light used to show fluorescence

describe the process of genetic fingerprinting

1. DNA is cut using restriction endonuclease

2. using electrophoresis separate DNA according to size

3. transfer to nylon membrane

4. make single-stranded an apply radioactive DNA probe