DNA technology

Use and steps of southern blotting:

Detect specific DNA sequences presence & type.

1. Separation by gel electrophoresis

2. Denaturation by NaOH

3. Blotting/transfer on nylon membrane or nitrocellulose paper

4. Hybridization

5. Detection

Use and steps of Sanger sequencing:

To identify base sequence in DNA.

It relies on chain termination using ddNTPs.

1. Fragmentation & amplification

2. Denaturation

3. Reaction mixture (primer + nucleotide bases + DNA polymerase +labelled ddNTPs)

4. Gel electrophoresis with polyacrylamide gel

5. Reading of sequence with automatic sequencer

Use, reagents & steps of PCR:

Advantages of real-time PCR over conventional PCR:

Goal/use of recombinant DNA technology

To produce clones of genetic material (genetic cloning)

Purpose & characteristics of a cloning vector:

To introduce foreign genetic material into cloning host.

Types of cloning vectors:

Steps of gene cloning experiment:

• Preparation & digestion of both chromosome (target DNA) & plasmid

• Ligation—→ recombinant or circularized plasmids

• Transformation

• Identification of recombinant plasmids bacteria (blue-white screening)

• Identifying colonies with desired gene (southern blotting)

• Amplification

First FDA approved, genetically-engineered drug by recombinant DNA technology is…………………..

insulin.

First FDA approved, genetically engineered vaccine was for……………………..

Hepatitis B.

Examples of genetically modified organisms:

• Modifying Pseudomonas syringae into an ice-minus bacteria strain (used in agriculture)

• Genetically modified K1026 strain of R. rhizogenes with modified K84 genes (forms AB to Agrobacterium tumefaciens, which causes crown gall disease in plants)

• Oncolytic viruses as the modified HSV against melanoma tumor cells.

Genetically-engineering plants depend on:

Agrobacterium tumefacien’s large plasmid, the Ti plasmid, with the region T-DNA.

Examples of transgenic plants:

1. Nicotiana tabacum (tobacco)= herbicide resistance

2. Pisum sativum (pea)= pest protection

3. Peanuts plant= have Bt genes for insect resistance

4. Oryza sativa (rice)= modified to produce beta-carotene (vit A precursor)

5. Cherry tomatoes with high levels of anthocyanin antioxidant

Transgenic animals production process is also called………………..

Germline-engineering.

Reproductive cloning is done through process called………………………

Somatic cell nuclear transfer (SCNT)

Genetic treatments include (with examples):

1. Gene therapy: Zolgensma for SMA ttt

2. ASO therapy: Tofersen for ALS

3. RNAi therapy: Patisiran for polyneuropathy

Techniques of functional genomics:

Function of DNA microarray:

to determine level of expression of thousands of genes simultaneously.

DNA microarray technique steps:

1. mRNA extracted—→reverse transcriptase—→cDNA

2. cDNA is labeled with fluorescent label

3. cDNA (probe/single strands) is put on DNA microarray (gene chip)

4. Hybridization occurs.

5. Unpaired cDNA are washed off

6. Scan with laser. Intensity of fluorescence is measure by bioinformatics to determine upregulated and downregulated genes

DNA-protein binding sites can be determined/studied by………………………..

Chromatin-immunoprecipitation (ChIP) assay.