Recombinant DNA Technology (cloning, libraries).pptx

Biology
Biotechnology

1. by Dr. Shaista Javaid Assistant Professor, Biotechnology Recombinant DNA Technology

2. How Do You Identify and Clone a Gene of Interest?

3. Creating DNA Libraries Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host Screened to pick out different genes of interest Two Types of Libraries Genomic DNA libraries Complementary DNA libraries (cDNA libraries)

4. Genomic Libraries Chromosomal DNA from the tissue of interest is isolated and digested with a restriction enzyme which produces many fragments that include the entire genome Vector is digested with same enzyme DNA ligase is used to ligate genomic DNA fragments and vector DNA Recombinant vectors are used to transform bacteria and theoretically each bacteria will contain a recombinant plasmid

5. Disadvantages of genomic libraries Introns are cloned in addition to exons; Majority of genomic DNA is introns in eukaryotes so majority of the library will contain non-coding pieces of DNA Many organisms have very large genome, so searching for gene of interest is difficult Time consuming!

6. cDNA Libraries mRNA from tissue of interest is isolated Need to make double stranded DNA from mRNA: How? enzyme reverse transcriptase catalyzes synthesis of complementary single stranded DNA from mRNA Called complementary DNA (cDNA) because it is an exact copy of the mRNA mRNA is degraded either with an enzyme or alkline solution DNA Pol is used to synthesize second strand of DNA to create double stranded cDNA Short linker double stranded DNA sequences which contain restriction enzyme recognition sites are added to the ends of the cDNA Cut with restriction enzyme , cut vector with same enzyme, ligate fragments to create recombinant vectors Then transform bacteria with recombinant vectors

7. cDNA Libraries Advantage over genomic libraries Collection of actively expressed genes in the cells or tissues from which the mRNA was isolated Introns are NOT cloned Can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue Disadvantage Can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available

8. Library screening to identify the gene of interest Colony hybridization Bacterial colonies containing recombinant DNA are grown on an agar plate Nylon or nitrocellulose filter is placed over the plate and some of the bacterial colonies stick to the filter at the exact location they were on the plate Treat filter with alkaline solution to lyse the cells and denature the DNA Denatured DNA binds to filter as single-stranded DNA Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye DNA fragment that is complementary to the gene of interest Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization

9. Why is it necessary to tag the probe with either a radioactive nucleotide or fluorescent dye that can be used to catalyze light releasing reactions?

10. Colony Hybridization Filter is washed to remove excess unbound probe Filter is exposed to film – autoradiography Anywhere probe has bound to the filter, radioactivity from the radioactive probe or released light (fluorescence or chemiluminescence ) from non-radioactive probes exposes silver grains in the film Depending on the abundance of the gene of interest there might be few colonies or plaques on the filter that hybridize to the probe Film is developed to create a permanent record of the colony hybridization Use digital instrument to detect probe binding if a fluorescent or chemiluminescent probe was used Film is then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest

11. Colony Hybridization Type of probe used depends on what is already known about the gene of interest Example: use mouse or rat probe to screen a human library because many genes between these species are similar If gene sequence has NOT been cloned in another species but something is known about the protein, what can be done? Library screening rarely results in the cloning of the full-length gene Usually get small pieces of the gene; the pieces are sequenced and scientists look for overlapping sequences ( contigs ) Look for start and stop codons to know when the full length of the gene is obtained

12. Polymerase Chain Reaction Developed in the mid-1980s by Kary Mullis Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time Process Target DNA to be amplified is added to a tube, mixed with nucleotides ( dATP , dCTP , dGTP , dTTP ), buffer, and DNA polymerase. Paired set of Forward and Reverse Primers are added – short single-stranded DNA oligonucleotides (20–30bp long) Primers are complementary to nucleotides flanking opposite ends of target DNA Reaction tube is placed in an instrument called a thermocycler

13. PCR Process Thermocycler will take DNA through a series of reactions called a PCR cycle Each cycle consists of three stages Denaturation – heat to 94 °C to 96 °C Annealing (hybridization) – in which primers H bond with complementary bases at the opposite ends of target sequence at 50 °C to 65 °C Extension (elongation) – DNA Pol copies target DNA at 70 to 75 °C At the end of one cycle, the amount of DNA has doubled Cycles are repeated 20–40 times

14. Advantage of PCR: Can amplify millions of copies of target DNA from small amount of starting material in short period of time To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation 2 N in which N represents number of PCR cycles Assume you want to do 22 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR?

15. The type of DNA polymerase used is very important Taq DNA polymerase – isolated from a species known as Thermus aquaticus that thrives in hot springs Why can't you use DNA Pol isolated from bacteria that live at 37 C? Applications Making DNA probes Studying gene expression Detection of viral and bacterial infections Diagnosis of genetic conditions Detection of trace amounts of DNA from tissue found at crime scene Detection of DNA from fossilized dinosaur tissue

16. Cloning PCR Products Is rapid and effective compared to using DNA libraries Disadvantage Need to know something about the DNA sequence that flanks the gene of interest to design primers Great trick: Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products Use this knowledge to researcher's advantage: T vector that has single stranded thymine on each end so can complementary base pair with the adenine in the PCR products