pcr and dna sequencing

POLYMERASE CHAIN REACTION (PCR): 

POLYMERASE CHAIN REACTION (PCR) Nobel prize winning discovery! Mr. Meghanath Prabhu Ph.D. Scholar BITS Pilani Goa Campus

The purpose of PCR is to: 

The purpose of PCR is to A) make more copies of DNA primers to increase protein synthesis B) make many copies of an organism’s DNA sequence so a small number of organisms will become large enough to be identified C) make more RNA so large units of protein can be synthesized D) recycle DNA using thermal cyclers

Reagents Required: 

Reagents Required DNA molecule having Target sequence Four nucleotides (dNTP’s) Primers DNA polymerase (Taq poly) Mg +2 Water!

Slide 5: 

The number of amplified pieces of DNA equals ____ after five cycles of PCR. A) 5 B) 10 C) 25 D) 32 Ans - D

Slide 6: 

Generally, PCR amplifies small DNA targets 100-1000 base pairs (bp) long. It is technically difficult to amplify targets >5000 bp long

For DNA amplification to occur, which of the following are needed?: 

For DNA amplification to occur, which of the following are needed? A) loose ribonucleotides B) RNA primers C) thermostable DNA polymerase D) b and c E) all of the above

Slide 8: 

Why do researchers use DNA polymerase from a bacterium found in superheated water for PCR? A. It's the only bacterium that contains this enzyme. B. It can withstand the elevated temperatures that are required to unwind DNA. C. DNA must go through high-temperature sterilization before PCR can occur. D. These bacteria contain more DNA polymerase than any other species. Ans-B

Taq polymerase starts copying at: 

Taq polymerase starts copying at A) the end of free single-stranded RNA B) any open point C) RNA primers attached to the end of the desired gene D) DNA primers attached to the end of the desired gene

Slide 10: 

Recently, the problem of infidelity of DNA replication during the PCR reaction has been considerably reduced by using alternative heat-stable DNA polymerases which have associated 3’ to 5’ exonuclease activity. Pyrococcus furiosus ( Pfu ) DNA polymerases and Thermococcus Litoralis (VENT) are becoming more widely used because of the proofreading conferred by their associated 3’ to 5’ exonuclease activity. Due to this Much lower level of mutations introduced by copying errors

Slide 11: 

Verification of PCR product

Things to remember: 

Things to remember

PRINCIPLE: 

PRINCIPLE Denaturation at 94°C, 1 min :the double strand melts open to single stranded DNA Annealing at 54°C, 45 sec : formation of hydrogen bonds between single stranded primer and single stranded bases. (Annealing temp . for primer = 4(G+C)+ 2(A+T) -5) Extension at 72°C, 2 min : after the primers attach the Taq polymerase begins to add nucleotide to form complementary strand

Applications of PCR:: 

Applications of PCR: Amplification of small amounts of DNA for further analysis by DNA fingerprinting. The analysis of ancient DNA from fossils . Mapping the human (and other species) genome. The isolation of a particular gene of interest from a tissue sample. Generation of probes : large amount of probes can be synthesized by this technique. Production of DNA for sequencing: Target DNA in clone is amplified using appropriate primers and then its sequence determined. Helpful in conditions where amount of DNA is small. Analysis of mutations : Deletions and insertions in a gene can be detected by differences in size of amplified product.

Applications of PCR: (cont..): 

Applications of PCR: (cont..) Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis, PCR is used to amplify DNA from foetal cells obtained from amniotic fluid. PCR has also proved very important in carrier testing. Detection of microorganisms : Especially of organisms and viruses that are difficult to culture or take long time to culture or dangerous to culture. The PCR has even made it possible to analyze DNA from microscope slides of tissue preserved years before. Detection of microbial genes responsible for some aspect of pathogenesis or antibiotic resistance. Crucial forensic evidence may often be present in very small quantities, e.g. one human hair, body fluid stain (blood, saliva, semen). PCR can generate sufficient DNA from a single cell.

Reading the blueprint of life: 

Reading the blueprint of life DNA sequencing The process of determining the order of the nucleotide bases along a DNA strand is called DNA sequencing

Introduction: 

Introduction The blueprint of life is contained in the DNA in the nuclei of eukaryotic cells and simply within prokaryotic cells. Human genome project – just obtain the list of approximately 3x10 9 bases (As, Cs, Gs and Ts) in the 23 chromosomes. Extraction of useful information from this list and genome sequence of other organisms relies on computer-intensive data handling – Bioinformatics.

Sequencing: 

Sequencing The DNA from the genome is chopped into bits- whole chromosomes are too large to deal with, so the DNA is broken into manageably-sized segments. The DNA is amplified by cloning into bacteria or by PCR It is then denatured (ie. melted), so that the two strands split apart.

Sequencing methods : 

Sequencing methods - In 1977 two separate methods for sequencing DNA were developed: the chain termination method or cycle sequencing or Sangers method ( Frederick Sanger et. al.) and the chemical degradation method or Maxam-Gilbert sequencing ( Allan Maxam and Walter Gilbert) - Both methods were equally popular to begin with, but, for many reasons, the cycle sequencing method is the method more commonly used today - This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis

Maxam and Gilbert Method: 

Maxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified

Maxam-Gilbert Technique: 

Maxam -Gilbert Technique 5’-CTTTTTTGGGCTTAGC-3’

Sanger Method: 

Sanger Method in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis

Dideoxynucleotide: 

Dideoxynucleotide no hydroxyl group at 3’ end prevents strand extension CH2 O O PPP 5’ 3’ BASE

The principles: 

The principles Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence

Slide 25: 

CCGTAC 3’ 5’ 5’ 3’ primer dNTP ddATP GGC A ddTTP GGCA T ddCTP GG C G ddGTP G G GGCAT G A T C G 5’ GGCATG 3 ’ complementary 3’ CCGTAC 5’

Slide 26: 

DNA sequencing gels are read from _____. A) the top down B) from backside using infrared light from the right side (when viewed from the loading wells) D) the bottom up from the left side (when viewed from the loading wells) Ans - D

Slide 27: 

Dideoxy nucleoside triphosphate _____. Has an oxygen at the 3' carbon Cannot bind to a growing DNA chain Can form a sugar phosphate bond with a new nucleoside triphosphate during DNA synthesis Has an oxygen at the 2' carbon E) Will terminate DNA synthesis when incorporated into a growing DNA strand Ans - E

Slide 28: 

In this gel, the base-pair lengths are listed to the right. Which end of the DNA fingerprint was plugged to the NEGATIVE terminal during electrophoresis? A) Top B) Bottom C) Right D) Left Ans- A

Comparison: 

Comparison Sanger Method Enzymatic Requires DNA synthesis Termination of chain elongation Maxam Gilbert Method Chemical Requires DNA Requires long stretches of DNA Breaks DNA at different nucleotides

Shotgun sequencing: 

Shotgun sequencing Shotgun sequencing dispenses with the need for mapping and so is much faster. It involves chopping the DNA into fragments of size c. 2000 base pairs (bps) and 10000 bps, sequencing the first and last 500 bps of each fragment. It then uses computer algorithms to assemble the entire sequence from the sequenced fragments. Working out DNA sequence ~ jigsaw puzzle

Lots of work to do…..!: 

Lots of work to do…..! What are different types of PCR? What is RT- PCR and R-T PCR?