How are genes cut?
- By restriction enzymes
- Bacterial enzymes that cut DNA in a sequence specific manner
What is a Plasmid?
A small double stranded circular DNA molecule that can replicate independently of the main bacterial chromosome. Can accumulate in hundreds of copies within each bacterial cell.
What must one do to study a gene in detail?
Isolate it from other genes of the organism and amplify it to obtain many identical copies
What are Restriction enzymes?
Restriction enzymes are enzymes that are bacterial in origin that can be used to cut DNA at specific sequences so it can be manipulated in the laboratory
What are Plasmids?
Plasmids are small double stranded DNA Molecules found in bacteria that cane replicated independently of the main chromosome (and can therefore accumulate in large number in cell)
What does Cloning involve?
Cloning involves cutting the DNA from the organism of inters with restriction enzyme and attach them to plasmids that have been opened at on location by a restriction enzyme. The joining is accomplished by DNA Ligase
How do you isolate and amplify a gene?
- After the DNA is cut by DNA Ligase and attached to plasmids the resulting plasmids a re then reintroduced into bacteria.
- The bacteria are spread on agate plates and allowed to grow to form colonies overnight.
- Each colony consists of millions of genetical identical bacteria all derived from single bacterial cell that started growing at that location the night before.
- All bacteria present in a colony contain the same cloned gene that is now isolated from all other genes in the cell.
What is the faster way to isolate and amplify a gene?
- A faster way to isolate and amplify a gene is with PCR.
- The target DNA is heated to separate the strands.
- Primers are then added along with DNA polymerase and four nucleotides.
- The reaction is cooled to allow the primers to bind and the DNA polymerase then copies of each of the strands
- Now you have 2 molecules insead of the 1 that you started with.
- Cycle is then repeated
- With each cycle, the amount of DNA doubles 1 to 2 to 4 to 8 to 16……
What is DNA Sequencing?
- FOur tubes labeled A,T,G,C
- Each tube gets the 4 normal nucleotides, DNA polymerase and single stranded DNA primer complementary to one end of the gene being sequenced.
- Each tube gets one type of Dideoxynucleotide (which can be incorporated into DNA but prevents the addition of subsequent nucleotide (stops DNA synthesis)
- Each Dideoxy nucleotide is tagged with a different color fluorescent tag.
- The A tube would generate different length DNA fragments all ending in A and all Fluorescing red.
- When the reactions are complete they are pooled and separated by size (small to largest).
- The order of colors indicter the orders of nucleotides in the DNA.
What does DNA sequencing due for us?
- Helps us identify new genes and their functions
- Revels stretches of DNA involved in the regulation of gene expression (Such as promoters and enhancers
How can DNA technology be used to solve crimes?
- The number of tandem repeated sequences differs at a number of chromosomal locations in each individual
- For this reason, when DNA is is cut with restriction enzymes it generates unique size fragments for each individual.
What are Histome proteins?
- Package the DNA so it fits in the Nucleus
- Keep the genes in an accessible state
- Repress gene expression
What is the Transcription factors?
- Proteins that bind to DNA and regulate gene expression
- Responsible for turning on and off genes (Opening certain his tomes)
What are the Enhancers with (TF)?
DNA sequences that stimulate gene expression via TF binding
What are the promoters?
Where RNA polymerase binds on DNA to initiate transcription
Why is one cell different from another?
What is a DNA chip?
A small piece of glass that contain many different genes 100,000 spots with different genes
What is Reverse transcriptiase?
- Comercial available
- Converts mRNA to cRNA
What is RNA splicing?
- Removing the interns from a strip of DNA
- Making mRNA
What are Primers?
Single stranded DNA complementary to the ends of the gene