Recombinant DNA Technology

• DNA replication refers to the scientific process in which a specific sequence of DNA is replicated in vitro, to produce multiple clones which can be used for a variety of purposes. There are two techniques that are used to replicate DNA and they are the use of bacterial plasmid vectors and a technique called polymerase chain reaction (PCR).  

• Bacterial plasmid vectors are used along with restriction enzymes to cleave the DNA at specific sequences, a DNA ligase to bind the recombinant DNA molecule together, and a bacterial cell to replicate the DNA.

OVERALL PROCESS

• The other technique, polymerase chain reaction (PCR) involves the use of heat to denature the double stranded DNA, and DNA primers which anneal to the original strand of DNA and add new, complimentary nucleotides to it, which yields a new double stranded DNA molecule.

• Replicated DNA can be used to in the laboratory to help catch criminals or it can be used to create transgenic organisms.  Organisms that contain DNA that is not from there original genome are called "transgenic" or "genetically modified". This technology is very useful in both plants and animals to create organisms that are stronger and more resistant to infections.

Transgenic Organisms

1. Use of RESTRICTION ENZYMES to cleave sections of long strand DNA into smaller, identifiable segments.

2. Use of MODIFICATION ENZYMES to combine “sticky ends” of segments together.

3. Use of CLONING VECTORS to reproduce modified segments.

Bacterial vector DNA replication process:

Restriction Enzymes

Restriction Enzyme ECO RI

Commonly used Restriction Enzymes

“Sticky ends”

“Blunt ends” vs. “sticky ends”

Cloning vectors – duplicates cleaved DNA

Advantages: Disadvantages:

• Replication errors are uncommon

• Used often in creating transgenic crops

• Takes a long time compared to PCR

• More expensive than other techniques

DNA Bacterial Vector replication

Most eukrayotic genes incorporate introns (non-coding sequences). Need to remove these introns before can exons can be read to produce new genes. Bacterial cells cannot read introns for vector replication.

REVERSE Transcriptase - Reading a protein to get a DNA sequence

1. Can circumvent this by using reverse transcriptase (from bacterial viruses). Catalyzes transcription in reverse. (Assembles complementary DNA strand on mRNA transcript).

Reverse Transcriptase & cDNA

PCR – Polymerase chain reaction

Quicker and faster than using vectors

A way to produce much MORE DNA or RNA

Steps:1.Purify DNA fragment that want to copy. 2.Heat DNA fragment to 92-94C (causes DNA to unwind)3.Add primers to DNA that base-pair with ends of fragment4.Primers acts as START for DNA polymerase to replicate DNA5.Let mixture cool6.Forms new DNA that coils7.Heat mixture again – causing DNA to unwind8.Repeat above steps. 9.Produces 1000’as DNA strands

PCR – Polymerase Chain Reaction

DNA Fingerprinting

Gel electrophoresis

DNA SEQUENCING

Automated, computerizedBased on ABSORBANCE of laser light due to differences in base structures

DNA sequencer readout

Creditshttp://www.personal.psu.edu/sjb316/blogs/engl202c/assignment-4.html