SBI4U0 Biotechnology

Polymerase Chain Reaction (PCR) Used to amplify a DNA sequence Each cycle only takes minutes, so after 20 cycles you have 1 048 576 copies Useful for forensic science, criminal investigations, medical diagnosis, genetic research Medical diagnosis  HIV viral DNA

PCR process 1. heat up DNA to break H bonds (94-96oC) to get 2 strands (serve as templates) 2. DNA primers used (made in lab) Forward primer and reverse primer 3. Drop temp down to 50-65oC so primers can anneal to template strands 4. Once primers anneal, Taq polymerase is added (DNA polymerase would denature).

Taq polymerase adds appropriate nucleotides starting at primer and stops at target region 5. After first cycle you end up with variable-length strands (mixture of DNA strands that have been replicated and are of unequal length). 6. Cycle repeats itself, makes constant length strands.

By the 3rd cycle the number of copies of the targeted strands begins to increase exponentially.

RFLP Restriction Fragment Length Polymorphism Polymorphism = any difference in DNA sequence (coding or non-coding) that can be detected between individuals (same genes but different alleles) Coding regions – can id people with specific mutations (i.e. Sickle cell anemia carries a different allele for -globin to give sickle shape

Non-coding regions – VNTR (variable number tandem repeats) aka microsatellites are used in forensic investigations RFLP analysis – DNA regions digested using restriction enzymes and radioactive complementary DNA probes are used to compare differences in DNA fragment lengths between individuals.

RFPL Procedure 1. DNA is extracted using restriction endonucleases and run on gel 2. Due to the large # of fragments they appear as a smear on the gel  placed against a nylon membrane and immersed in denaturing solution to break into single stranded DNA 3. single stranded DNA migrates from gel to nylon membrane (+) using electric current (SOUTHERN BLOTTING)

4. The nylon membrane is now soaked in a solution containing radioactive probes. The radioactive probes bond to complimentary base pairs at a specific region (hybridization) 5. The nylon membrane is put against x-ray film for 2-3 weeks and the radioactive probes burn an image into the x-ray film (audiogram)

6. The film is developed and the pattern is detected.

Coding regions

Non-coding regions

Summary State of sample RFLP PCR State of sample Large and fresh (blood size of quarter, semen size of dime) Minute and degraded (one cell) Size Whole genome Target sequence suffices Time 3 weeks (avg) One day Basic Premise Cleaving DNA using restriction enzymes followed by subjection to radioactive complementary DNA probes Building complementary strands using principles of DNA replication

Sensitivity and accuracy Highly sensitive and accurate RFLP PCR Result medium Autoradiogram Gel Tools Restriction enzymes, radioactive probes, nylon membrane, x-ray film, gel electrophoresis DNA polymerase, nucleotides (A,G,C,T), DNA primers, gel electrophoresis Sensitivity and accuracy Highly sensitive and accurate Sensitive and accurate

To analyze gene structure and its relationship to gene expression and protein conformation. Sanger dideoxy method: DNA sequencing technique based on DNA replication that uses dideoxy nucleoside triphosphates Uses the process of DNA replication Dna sequencing

Dideoxy nucleoside triphosphate Dideoxyanalogy  sugar is missing OH group on 3’ carbon

Frederick Sanger Developed method in 1977 Sanger and colleagues were the first to sequence an entire genome Bacteriophage (viral DNA) – 5386 base pairs in length

Sanger dideoxy method 1. DNA template to be sequenced is treated so that it becomes single stranded 2. radioactive primer is added to the end of the DNA template 3. Identical copies of primed single stranded DNA is placed in 4 reaction tubes.

4. Each tube contains DNA polymerase and a supply of free nucleotides (dATP, dTTP, dGTP, dCTP). Each tube also has a different radioactive dideoxy analog of one of the dNTP Tube 1 – all 4 bases + ddATP Tube 2 – all 4 bases + ddTTP Tube 3 – all 4 bases + ddGTP Tube 4 – all 4 bases + ddCTP

5. DNA polymerase binds and builds a complementary DNA strand Since the ddNTP is missing the OH on the 3’ carbon, DNA polymerase can’t bind  chain termination Only a small amount of dideoxy analogs are present in each tube, so the new complementary strands are built to different lengths

3’ – AATGCATGCATTAGC – 5’ 5’ - TTA TTACGTA TTACGTACGTA TTACGTACGTAA – 3’

Gel placed against x-ray film, and radioactive ddNTP expose the film 6. Since the strands are of different lengths, they can be separated using gel electrophoresis. Contents of each of the reaction tubes loaded into 4 different lanes of the gel Fragments differ only in 1 bp, so it can be read right from the gel Gel placed against x-ray film, and radioactive ddNTP expose the film

Human Genome Project Uses the Sanger dideoxy method Difference – ddNPT are fluorescently tagged E.g. ddGTP – green ddATP – yellow Multiple computers were working to read the sequence and position on the gel to determine the code Human genome is 3 billion base pairs long