2 Recombinant DNADNA that is artificially made with specific gene sequences added to itTo insert a gene, you must:Use restriction enzymes (restriction endonucleases) to cut up DNA at specific base sequences (figure 20.3) from both the source of the gene and the destination DNAThis creates “sticky ends” of DNA fragments; DNA sequences cut with the same restriction enzymes can be joined together (will use complementary base pairing rules)DNA ligase can seal the sugar-phosphate backbone together so the foreign DNA is now included = RECOMBINANT DNA
3 Recombinant DNARecombinant DNA can be inserted into bacterial cells by making plasmids (small circular segments of DNA), which bacteria will take up – this is called bacterial transformation (we will do this in AP Lab 8 – insert fluorescence gene from jellyfish into bacteria)A modified plasmid that accepts foreign DNA is called a cloning vector; bacteria can then be used to make large quantities of a desired protein (such as human insulin)See figure 20.4 on page 399 for diagram of cloning genes in bacterial plasmids
5 cDNAcomplementary DNA, made from backwards transcription (mRNA DNA) – fig. 20.6When foreign genes are inserted into a bacterial plasmid with recombinant DNA technology, introns can prevent transcription.Thus, scientists instead use a mature mRNA transcript (with introns already cut out) and use an enzyme called reverse transcriptase to build a complementary DNA (cDNA) from the mRNA.cDNA can then be inserted into a plasmid and bacteria are able to make the protein of interest.
6 Gel electrophoresisprocess by which restriction fragments of DNA (cut by various restriction enzymes) are separated in a gel (see fig. 20.9)DNA fragments diffuse through a jello- like material (agarose gel) that has been placed in an electric field.DNA is negatively charged, so the DNA fragments will move toward the positive end.Shorter DNA fragments will migrate faster through the gel’s pores than longer, heavier fragments.
7 Gel electrophoresisGel electrophoresis can then be used to compare DNA fragments of closely related species to determine evolutionary relationships, or to compare individuals of the same species (crime scene analysis, paternity testing, etc.)In these cases, the fragments differ in length because of polymorphisms –slight differences in DNA sequences, called RFLPs (used in DNA fingerprinting)RFLPs – Restriction Fragment Length Polymorphisms; SNP that exists in the restriction site for a particular enzyme = site is unrecognizable by enzyme and changes the length of fragments in digestion of DNA (coding or noncoding DNA); used to find differences among individuals based on varying lengths of fragments (varying alleles)
8 SNPs – Single Nucleotide Polymorphisms; occur 1 in base pairs; single base-pair site in a genome where variation occurs in at least 1% of the population (coding or noncoding DNA)
9 VNTRsVariable Number Tandem Repeats (also calleds STRs—short tandem repeats)A short nucleotide sequence is repeated in varying amounts in different individualsThey can be separated from surrounding DNA by RFLP or PCR methods and use gel electrophoresis or Southern blotting to determine sizeThe banding pattern found is unique to each individual (the same banding pattern is unlikely in 2 unrelated individuals)Used in forensics
10 PCR (polymerase chain reaction) Method used to amplify a piece of DNA without using cells containing the DNA; used when the DNA source is impure or scant (like a crime scene) – see fig 20.8Cycle #1 = 2 DNA moleculesHeat piece of DNA to separate 2 strands (denaturation)Cool to allow mRNA primers to form H-bonds with ends of DNA (annealing)DNA polymerase adds nucleotides to 3’ end of primers (extension)Cycle #2 = 4 DNA molecules; repeat steps 1-3Cycle #3 = 8 DNA molecules; repeat steps 1-3**This process provides ample DNA to analyze.**PCR animation Scientists for a better PCR
12 Nucleic Acid Hybridization Used to find the gene of interest among many colonies after transformationIf you know part of the gene sequence you are looking for, you can make the complementary piece (and make it radioactive as a tracer) to guide you to the proper gene of interest
13 Southern Blottingcombines gel electrophoresis and nucleic acid hybridization to find a specific human gene; can find differences between alleles (see fig )For example, it can distinguish between normal hemoglobin gene and one for sickle cellUse gel electrophoresis to look at homozygous dominant and recessive alleles and compare it to individual’s DNA. You can determine whether it is homozygous or heterozygous using the gelUse nucleic acid hybridization by making a radioactive single stranded DNA molecule that is complementary to gene of interest (i.e.- sickle cell)See Fig in book for full diagram and description
15 DNA microarray assay Enables genome-wide gene expression studies Small amounts of single-stranded DNA fragments are placed on a glass slide (DNA chip)mRNA molecules are isolated and converted to cDNA (reverse transcriptase) and tagged with fluorescent dye)cDNA bonds to the single-stranded DNA on the chip, showing which genes are “on” and producing mRNA by the location of the dye (for example breast cancer tumors vs. noncancerous breast tissue)See Fig in book for full diagram and description
17 Other practical DNA technology applications: Human Genome Project: global effort completed to sequence all of the DNA base pairs in the human genome (3 billion base pairs!); using the information to advance medical treatmentsStem cells: have enormous potential for medical applicationsGene therapy: alteration of an afflicted individual’s genes – can be used to treat single gene disorders such as cystic fibrosis or SCID
18 Other practical DNA technology applications: Environmental cleanup: genetically engineered microorganisms used to treat environmental problems such as removing heavy metals from toxic mining sites or cleaning chemical spillsAgricultural applications: genes that produce desirable traits are inserted into crop plants to increase their productivity or efficiency (called GMOs – genetically modified organisms); ex. golden rice