Recombinant DNA

What are the base pairs?

A-T
G-C

What is artifical selection/ selective breeding?

Selecting which male and female organisms are crossed to produce an offspring in order for certain, desired genes to be present in DNA in the next generation (increasing cahcne of phenotypes to be passed on)
-This is a slow and inefficient process

What is genetic engineering/ recombinant DNA technology?

Involves the atrifical modification of DNA, where DNA is either added or removed from a cell.
The DNA produced is called recombinant DNA and the organisms is a genetically modificied organism.

What is recombinant DNA technology used for?

Introducing genes for desired traits into organisms, using harmless bacteria to produce proteins and replacing faulty genes.

What is a transgenic organism?

When DNA from one species being introduced into a different species, the organism produced is a transgenic organism
Transgenic organism introduce a trait not normally present

What is restriction enzyme?

Naturally occuring enyzme found in bacteria that can cut parts of DNA at a specific base sequence (called recognition site)

What is the recognition site?

A specific sequence of DNA that is recognized and cleaved by a restriction enzyme. These sites, typically 4-8 base pairs long, are essential for creating DNA fragments
-They're palindromic meaning they ahve the same sequence when read forwards and backwards.

What is a straight cut?

When the resirtcition enzyme makes a clean break across the two strands of DNA to produce a blunt end (cuts both DNA strands at the same location)
-Blunt end is when both strands terminant in a base pair

What is a blunt end?

Both DNA strands terminate in a base pair (strands are equal length), meaning no overhangs are present

What is a staggered cut?

Results in fragements with stick ends (single-strand overhangs)

What is a sticky ends?

A sticky end is a stretch of unpaired nucleotides in a DNA molecule that overhang at the break in the strands.
They have the ability to combine with sections of DNA that have a complementary ending (useful in recombinant DNA technology as allows for one DNA fragment to be paired with other DNA fragment with a corresponding sequence)

What is DNA ligase?

Can join, recombine or seperate parts of DNA.
Used in recombinant DNA to "gluse" together short strads of DNA during replication (process called ligation)

How does DNA ligase work?

Joins the phosphate group at the end of one strand to the sugar molecule at the end of another strand. The complementary bases must first join by forming hydrogen bonds and then the DNA ligase can join the backbone of each strand.

What is the first step of making recombinant dna (isolation of the gene of interest)

The gene of interest (e.g. insulin or antigen) is isolated (usually isolated from a human donor organisms DNA).

What is the second step of making recombiant DNA (cutting DNA with restriction enzymes)?

The restriction enzyme cuts the DNA at a specific recognition site of either side of the gene of interest
This creates creates stick ends (stretch of unpaired nucleotides in a DNA molecule that overhang at the break in the strands.)

What is step 3 of making recombiant dna (plasmid vector)?

The plasmid (a circulator DNA molecule which acts as a vector) is isolated from bacterial cell.
The same restriction enzyme is used to cut the plasmid at the restriction site to produce complimentary stick ends to the gene of interest on the plasmid

What is a plasmid?

Circular, double-stranded units of cytoplasmic DNA, frequently found in bacteria, which is capable of replicating within a cell indepdently of the chromosomal DNA.

What is step 4 of making recombiant dna (insertion)?

The gene of interest is now inserted into the cut plasmid. The enzyme DNA ligase joins the sticky ends (one from gene and one from plasmid)
This forms recombinant DNA which is the name given to any DNA that has been artifically made by joining components off diffferent organisms.

What is step 5 of making recombiant dna (cloning)?

The recombinant plasmid is then introduced into the bacterial cell (now called a transgenic organisms- living thing that contains genes of different organisms).
Replication transgenic organism occurs, causing the foregin gene to be clone. This is so there is numerous copies of the DNA available to insert into the host cell.

What is step 6 of making recombiant dna (replication)?

Once a large quantitiy of transgenic organism has been produced, it is introduced to the selected host cell such as special bacterial, yeast or mammalian cells. These then grown to multiply/ clone/ reproduce and produce the foregin protein using instructions in the gene in the recombinant DNA

What is the final step of recombinant DNA?

The host bacterial cell uses the inserted gene of interest to produce the desired protein (e.g., insulin). This protein is then harvested, purified, and prepared for use—such as for medical treatment in humans.

What are some recombinant vaccines already existing?

Hepatitis B
Human papilloma virus (HPV)
In recombinant vaccines, the antigen is produced and then introduced to the vaccine

What is DNA vaccines?

The DNA for the antigen is introduced in the vaccine instead of the antigen itself. This means the DNA is incorporated into the host's cells which then produce the antigen and will be expressed by host cells (similar ways to what happens in a viral infection)

Why does the development of recombinant DNA vaccines have disadvantage?

1. Very expensive: Genes for desired antigens most be located, clone and expressed by new vector
2. Research must be conservative