2 DNA SequencingDNA sequencing determines the exact sequence of base pairs for a particular DNA fragmentthe first method to sequences DNA was called chain terminatingit involved replicating DNA using chain terminating nucleotides that created small DNA fragments.the fragments are then run on a gel matrix.
3 this method was developed by Fredrick Sanger, Alan Maxim and Walter Gilbert variations of this method was used by the human genome project.
5 The Human Genome Project Started in 1990 and finished in 2008 (James Watson!)Estimates 3 billion base pairs of nucleotides.The haploid human genome contains ~23,000 protein-coding genes, far fewer than had been expected before its sequencing. In fact, only about 1.5% of the genome codes for proteins, while the rest consists of non-coding RNA genes, regulatory sequences, introns, and (controversially named) "junk” DNA.Aim of the project: new drugs and genetic therapies to combat genetic diseases such as CF, Huntington, etcEarlier predictions estimated that human cells have as much as 200,000 genes
6 DNA Sequencing Recombinant DNA fragment of DNA composed of sequences originating from at least two different sources.Genetic Transformationintroduction and expression of foreign DNA in a living organism.
8 Tools of Biotechnology Restriction Endonucleasesalso called restriction enzymesact like molecular scissors cutting double-stranded DNA at a specific base pair sequenceeach type of restriction enzyme recognizes a particular sequence of nucleotides.this is known as the recognition site.
9 molecular biologists use these enzymes to cut DNA in a predictable and precise way. most recognition sites are four to eight base pairs long and are usually characterized by a complementary palindromic sequence.example EcoR1Enzyme Source Recognition Sequence CutEcoR1 Escherichia coli 5'GAATTC 5’…G AATTC…3'3'CTTAAG 3’…CTTAA G…5'
10 EcoR1 scans the DNA molecule and binds at its recognition site. once bound it cuts between the Adenine and Guanine nucleotide on each strandat the end of each cleavage site one strand is longer than the otherit has exposed base pairs that lack complementary base pairs.these are called sticky ends.
12 not all restiction enzymes produce sticky ends. Enzyme Source Recognition Sequence CutSma1 Serratia marcescens 'CCCGGG 5'---CCC GGG---3' 'GGGCCC '---GGG CCC---5'the ends of the DNA fragments are fully base paired.these ends are called blunt ends.blunt ends have no sticky ends.
13 restriction enzymes that produce sticky ends are generally more useful as a biological tool. sticky ends can be joined to other DNA fragments created by the same restriction endonucleasemolecular biologists select restriction endonucleases that cut around genes of interestrestriction enzymes are named according to the bacteria they come from.first letter is the genus namesecond and third letter are the initial letters of the species name.fourth letter indicates the strainnumbers represent the order of discovery.
14 how many fragments will be produced is Sma1 digests this sequence? 3 5'-AATTCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTAGCA-3'3'-TTAAGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAATCGT-5‘how many fragments will be produced is Sma1 digests this sequence?3What types of ends does Sma1 produce?Blunt
15 Methylasesenzymes that can modify a restriction enzyme siteadds a methyl (-CH3) group to one of the bases in the sitethey protect a gene fragment from being cut at an undesired locationIn bacteria, it’s a crude type of immune system that was observed to prevent E.coli from being infected by viruses.In eukaryotes, usually occurs to inactivate a specific gene.
16 Ligasesto create recombinant DNA, pieces of DNA from two sources must be joined together.using restriction enzymes and methylases, geneticists can engineer fragments of DNA with desired genesthese fragments are joined together by DNA ligase.DNA ligase joins sticky ends together.
21 Taq DNA Polymerase and the Polymerase Chain Reaction in 1985 Kary Mullis invented the technique called the polymerase chain reaction (PCR)PCR allows scientists to make billions of copies of pieces of DNA from extremely small quantities.the reaction depends on the Taq DNA polymeraseTaq DNA polymerase synthesises DNA during replicationworks at very high temperatures because the bacteria it comes from lives in high temperatures (thermophile)each cycle of PCR doubles the number of DNA molecules.
22 molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8TECHNIQUE53Target sequenceGenomic DNA351Denaturation53352AnnealingCycle 1 yields2moleculesPrimers3ExtensionNew nucleo- tidesFigure 20.8 The polymerase chain reaction (PCR)Cycle 2 yields4moleculesCycle 3 yields 8molecules; 2 molecules (in white boxes) match target sequence
23 TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a Figure 20.8 The polymerase chain reaction (PCR)
26 molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8dCycle 3 yields 8molecules; 2 molecules (in white boxes) match target sequenceFigure 20.8 The polymerase chain reaction (PCR)
27 Transformation of Bacteria Genes are isolated and then inserted into a plasmid.Plasmids are circular double stranded DNA that occur naturally in many bacteria.Homework: Read pages of your textbook and then answer question 8, due tomorrow.