DNA technology

What is bioinformatics?

The storage and analysis of large datasets relating to biological material (DNA sequences, protein sequences etc) 

Is eukaryotic easy or difficult to sequence?

In eukaryotes, sequencing the genome (all of the genetic material of a cell/organism) does not allow you to easily predict the proteome (all of the proteins that can be produced by the cell/organism). 
This is because: 
a) Much of the DNA is non-coding (98% in humans) 
b) Many of the genes are not being expressed – just because the gene is there doesn’t mean that the protein is there too 

What is genetic engineering?

DNA from one organism is isolated and then placed into/combined with the genome of another organism, also called recombinant DNA technology. Leads to transgenic organisms or genetically modified organisms (GMOs) 

What is the Human Genome Project?

It is an international scientific project which has successfully determined the sequence of bases of a human genome, running from 1990-2003

Key steps in genetic engineering

1. Extraction of DNA e.g. using detergent to break down the phospholipid bilayer of the plasma membrane and nuclear membrane and release the DNA  

2. Isolation of the gene – restriction endonucleases are used to cut out the gene from the human DNA/genome. These enzymes cut at very specific locations 

3. Insertion of gene into a vector – the vector (e.g. a plasmid from E Coli) is cut open using the same restriction enzyme (to have complementary sticky ends and allow hydrogen bonds to form) and then the gene is inserted into the vector using the enzyme DNA ligase. DNA ligase catalyses the formation of a phosphodiester bond between the gene and the vector. This makes recombinant DNA 

4. Transformation – the recombinant DNA is transferred into a host cell (e.g. the E. coli is treated with heat or chemical shock to open up the membrane). In a tissue culture there is the issue that not all cells will become successfully transformed 

5. Identification – check cells have been successfully transformed using gene markers e.g. antibiotic-resistant gene. Cells that have transformed successfully will have both the insulin gene and the antibiotic resistance gene, as they are in the same plasmid. Those that did not successfully transform will not have the insulin gene or the antibiotic resistance gene because they did not take up the plasmid (which contains both genes). When introduced to antibiotic, only the cells that did not transform effectively will be killed (could alternatively use a fluorescent gene rather than antibiotic resistant) 

6. Growth/cloning: the transformed cells are cloned. Secreted insulin is collected and purified (issues of binary fission is that not all daughter cells may have the plasmids – however rapid cell division decreases this frequency) 

Restriction endonucleases

• Found naturally in bacteria, helps bacteria to protect themselves against foreign viral DNA (bacteriophages) 

• Cut DNA at very specific sequences  

• Different restriction enzymes cut at different sequences 

• e.g. EcoRI cuts ONLY at GAATTC, BamHI cuts only at GGATCC, which is palindromic  

• Most restriction enzymes make a staggered cut in the two chains, forming sticky ends. Sticky ends are only complementary to ends that have been cut by the same restriction enzyme. However, some restriction enzymes cut straight across both chains, forming blunt ends 

Issues of genetic engineering

• Genes of bacteria don’t have introns, therefore do not have sufficient enzymes to splice introns out of human genes that are inserted into plasmids. The introns will not code correctly for the right protein, as it cannot recognise introns.  
  Mature insulin mRNA is extracted from cells (meaning only exons and no introns) and copied to make single-stranded complementary cDNA using the enzyme reverse transcriptase. DNA polymerase is then used to make a double stranded version of the cDNA. DNA produced from the mRNA is put into the bacteria, as it then has no introns 
  - Reverse transcriptase makes single stranded cDNA from mature mRNA 
  - DNA polymerase synthesises second DNA strand to the cDNA 
  Disadvantages of this method as that mRNA can only be taken from a cell that is actively making insulin, the pancreatic cells. Single stranded mRNA is also vulnerable to degradation, so it is difficult to work with. 
  Alternatively, you can chemically synthesise the gene you want (including missing out the introns) 

• Prokaryotic cells don’t have membrane-bound organelles, for example a Golgi body, which is important for protein secretion 

How to chemically synthesise a gene

The ‘gene machine’

• Work backwards from the desired protein primary structure 

• Input desired base sequence for amino acids into a computer  

• Can’t build a whole gene in one go, so instead short overlapping sections of nucleotides (oligonucleotides) are chemically built 

• These then join together 

• Thousands of copies are made  

• These can then be inserted into the plasmid (by adding restriction enzyme sites on the ends of the gene) 

Origin of replication

where plasmids start DNA replication. Ensures the plasmid replicates quickly to increase the likelihood of daughter cells inheriting the plasmid 

Selectable marker

fluorescent gene used to reduce risk of false positives  

Promoter

the promoter region must be ‘upstream’ of the gene. It allows RNA polymerase and transcription factors in the host cell can bind and transcribe the gene into mRNA

What are three reasons why a host cell might not take up a recombinant plasmid?

1. The recombinant plasmid doesn’t get inside the cell 

2. The plasmid re-joins before the DNA fragment entered  

3. The DNA fragment sticks to itself, rather than inserting into the plasmid 

What are two ways in which you can identify if a cell has transformed correctly and successfully uptaken the recombinant plasmid?

Use of marker genes on the plasmid

• antibiotic resistance genes (replica plating)

• genes coding for fluorescent proteins (GFP) or coding for enzymes

Overview of replica plating as a method of identification

• plasmid will include two different antibiotic resistance genes e.g. ampicillin and tetracycline

• the gene of interest is inserted inside the resistance to tetracycline gene and disrupts it. Therefore this gene will no longer create a functional protein (although will still be resistant to ampicillin) 

• bacteria that are able to grow on agar with ampicillin have uptaken a plasmid, as they must contain an ampicillin-resistance gene (however the plasmid may not necessarily be recombinant and have the gene of interest)

• transfer some of the bacteria to agar with tetracycline. Any colonies still growing must contain bacteria without the gene of interest, as there is no DNA fragment to disrupt the tetracycline resistance gene. Bacteria resistant to ampicillin but not tetracycline contain the recombinant plasmid  

How do fluorescent markers work as a method of identification?

• Jellyfish contain a gene which codes to create a green fluorescent protein. Plasmid created with GFP gene in  

• DNA fragment (insulin gene) is inserted into the middle of the GFP gene. This disrupts it and prevents GFP production 

• Grow the bacteria on agar. When exposed to UV light, the ones with GFP gene intact glow. Therefore, only non-glowing colonies contain the recombinant plasmid 

What is the Polymerase Chain Reaction

• PCR is a commonly used technique in molecular biology to make millions of copies of a stretch of DNA of interest  

• In vitro DNA replication  

• It amplifies ‘target DNA’ (without amplifying DNA that you’re not so interested in) Amplify = copy and replicate 

• It uses DNA polymerase  

• Takes place inside a machine called a thermocycler 

Components of PCR

• DNA sample containing the target DNA 

• Taq DNA polymerase: DNA polymerase that has been made by bacteria rather than humans (from bacterium Thermus aquaticus, which is found in hot springs and therefore has an optimum temperature of 72 degrees. Ensures the DNA polymerase doesn’t denature) 

• DNA primers: artificial complementary sequences of DNA that latch onto the ends of the desired sequence so that DNA polymerase can bind   

• Free nucleotides (an excess) 

• (pH buffer)

• (Cofactors