B2. Making DNA Fragments

Question 1

Recombinant DNA technology

What does Recombinant DNA technology involve?

What two things allow transfer of DNA to produce proteins in the cells of the recipient organism?

What does one of those things allow to happen?

What are transgenic organisms?

Answer 1

• Recombinant DNA technology involves transferring a fragment of DNA from one organism to another.
• Because the genetic code is universal and because transcription and translation mechanisms are pretty similar too, the transferred DNA can be used to produce a protein in the cells of the recipient organism.
• The recipient and donor organisms don’t even have to be from the same species. This can be pretty useful.
• Organisms that contain transferred DNA are known as transgenic organisms

Question 2

Methods for making DNA fragments
In order to transfer a gene from one organism to another, you first need to get a DNA fragment containing the gene you’re interested in (the target gene). There are three ways that DNA fragments can be produced: (3 ways)

Answer 2

Method 1 - using reverse transcriptase

Method 2 - using restriction endonuclease enzymes

Method 3 - using a gene machine

Question 3

Method 1 - using reverse transcriptase

What makes it difficult to obtain a DNA fragment?

Why is mRNA often easier to obtain?

What can mRNA molecules be used for?

What does reverse transcriptase do?

3 steps

Answer 3

Method 1 - using reverse transcriptase

• It is difficult to obtain a DNA fragment containing the target gene because most cells only contain two copies of each gene.
• mRNA is often easier to obtain because cells that produce the protein coded for by the target gene will contain many mRNA molecules that are complementary to the gene
• The mRNA molecules can be used as templates to make lots of DNA.
• The enzyme, reverse transcriptase, makes DNA from an RNA template making complementary DNA (cDNA)

1. To make cDNA, mRNA is first isolated from cells.
2. Then it’s mixed with free DNA nucleotides and reverse transcriptase.
3. The reverse transcriptase uses the mRNA as a template to synthesise new strands of cDNA

Figure 1: cDNA synthesis using reverse transcriptase.

Question 4

Tip: Remember that DNA is copied into mRNA during _______________

Answer 4

Tip: Remember that DNA is copied into mRNA during transcription

Question 5

Tip: The cDNA is a _______________copy of the mRNA because of _________ _____ ________

Answer 5

Tip: The cDNA is a complementary copy of the mRNA because of specific base pairing

Question 6

Method 2 - using restriction endonuclease enzymes

Some sections of DNA have…. that consist of…

What do restriction endonucleases do? (3)

2 Steps

Answer 6

Some sections of DNA have palindromic sequences of nucleotides. These sequences consist of antiparallel base pairs

Restriction endonucleases:

• They are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places.
• Different restriction endonucleases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzyme’s active site
• You can use restriction endonucleases to separate it from the rest of the DNA if recognition sequences are present at either side of the DNA fragment you want

1. The DNA sample is incubated with the specific restriction endonuclease, which cuts the DNA fragment out via a hydrolysis reaction. Sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of the fragment.
2. Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences

Figure 2: A palindromic DNA sequence.
Figure 3: Using a restriction endonuclease enzyme to cut DNA.

Question 7

Tip: Using restriction endonucleases produces fragments of the whole bit of DNA you want including the _______. However _________ _____________ uses _______to make DNA so you only get _____

Answer 7

Tip: Using restriction endonucleases produces fragments of the whole bit of DNA you want including the introns. However reverse transcriptase uses mRNA to make DNA so you only get exons

Question 8

Exam Tip
Make sure you use the right words to describe these processes in the exam, e.g. _______________ _________not ‘the same shape.

Answer 8

Exam Tip
Make sure you use the right words to describe these processes in the exam, e.g. complementary shape not ‘the same shape.

Question 9

Method 3 - using a gene machine

More recently, technology has been developed so that fragments of DNA can be synthesised from scratch, without the need for a pre-existing DNA template. Instead, a database contains the necessary information to produce the DNA fragment. This means that the DNA sequence does not have to exist naturally any sequence can be made.

How is it done? (4 steps)

Answer 9

1. The sequence that is required is designed (if one doesn’t already exist).
2. The first nucleotide in the sequence is fixed to some sort of support e.g. a bead.
3. Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups.
   Protecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching.
4. Short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. Once these are complete, they are broken off from the support and all the protecting groups are removed. The oligonucleotides can then be joined together to make longer DNA fragments.