Gene Technology

Question 1

State the three methods for obtaining DNA fragments

Answer 1

Using restriction endonuclease
Using reverse transcriptase
Using a ‘gene machine’

Question 2

Describe how restriction endonuclease can obtain DNA fragments

Answer 2

Group of enzymes that n aurally occur in bacteria
They cut DNA at a specific sequence of bases (called a recognition sequence) by hydrolysing the phosphodiester bonds in the sugar phosphate backbone
They cut the DNA between opposite base pairs in a staggered structure. This creates sticky ends that can bind to other sticky ends with complimentary base pairing

Question 3

What are sticky ends?

Answer 3

Single stranded sections of DNA that form an overhang at the end of a double stranded molecule

Question 4

How is a gene machine used to obtain fragments of DNA?

Answer 4

Where genes are synthesised artificially:
- the amino acid sequence of a gene is determined
- the mRNA codons for each amino acids are searched
- the complimentary DNA triplets for the mRNA sequences are worked out and the gene is produced
-the genes are checked using standard sequencing techniques. Any with errors are rejected

Question 5

What are benefits of producing artificial genes with the gene machine?

Answer 5

Genes are free of introns and other non-functioning DNA- so they can be transcribed and translated

Question 6

After isolation of fragments of DNA, how is the number of DNA fragments increased?

Answer 6

Polymerase Chain reaction

Question 7

What are primers?

Answer 7

A short sequence of single stranded nucleotides, that have a specific base sequence

Question 8

What are the components of a thermal cycle used in PCR?

Answer 8

DNA fragments that need to be copied
Taq polymerase
Free nucleotides
Primers

Question 9

Explain the process of the polymerase chain reaction

Answer 9

In the thermal cycler, at 95*C the hydrogen bonds are broken and DNA double strand separates.
Temp is reduced to 55, primers attach to complimentary base sequences at the ends of the single strand
DNA polymerase attaches and the temp is raised to 72 (optimum temp for enzymes)
Free nucleotides attach to the single stranded templates via complimentary base pairing
DNA polymerase joins nucleotides together with phosphodiester bonds
DNA doubles in quantity