Unit 2a Molecular Biology Techniques Key Terms The Central Dogma In situ hybridization immunostaining GFP fusion protein confocal microscopy RNA overexpression RNAi morpholino antisense oligonucleotides morpholinos Transgenics knockouts homologous recombination Maternal effect mutants zygotic mutants Learning Objectives By the end of this unit you should be able to 1 Define the central dogma and explain why studying the function of a protein can often be performed by manipulating the gene or mRNA that encodes the protein 2 Explain what information in situ hybridization provides and be able to interpret the results 3 Explain what information immunostaining provides and how is it different from in situ hybridization 4 Briefly describe how a fusion protein is made and why tagging a protein with GFP is a useful tool for developmental biologists 5 Explain why confocal microscopy is especially useful for studying fluorescent molecules within embryos Note you do not need to explain the detailed operation of a confocal microscope 6 Explain in general what information mRNA overexpression provides 7 Explain in general terms two different methods of knocking down gene expression 8 Explain in general terms how transgenic embryos are generated 9 Explain in general terms how to generate a knockout mouse using homologous recombination and how selectable markers are used to isolate knockout ES cells as part of this procedure 10 Explain in general terms how mutants are generated and be able to explain the difference between a zygotic mutant and a maternal effect mutant 11 Explain the difference between forward and reverse genetics and give an example of each 12 For each of the molecular techniques covered in class you should be able to state the Big Idea as stated in the handout and be able to select a particular technique to answer a particular question about a developmental biological process 13 Important note You will NOT be asked to explain or use many techniques listed in this handout You are only responsible for those covered in class This handout is provided for the purpose of completeness and as a future reference for students performing developmental biology research in a laboratory setting Reading assignment The Big Ideas in this section Please see the following web page http 10e devbio com chapter php ch 2 Select 2 3 Techniques of DNA Analysis and 2 4 Techniques of RNA analysis Note there is much more detail than we will cover in class in these readings from Gilbert Molecular Techniques in Developmental Biology Big idea DNA encodes RNA which after appropriate processing produces a messenger RNA mRNA mRNAs in turn are translated into proteins Thus by working with DNA we are working with information required to make a protein DNA is a very stable molecule that has convenient properties we can take advantage of RNA on the other hand is highly unstable and tends to degrade rapidly and proteins can only be studied biochemically Molecular biology is the powerful technology that allows us to work with DNA and RNA and proteins using a number of different techniques Campbell 5e Fig 17 3 Electrophoresis Big idea Separate proteins or nucleic acids DNA RNA by size by driving them through a gel made of a polymer like polyacrylamide or a gel like agarose Why do this Since DNA and RNA are made of four simple building blocks in long chains we can t tell them apart chemically It s the number of bases and their order that matters This technique at least allows us to distinguish different DNAs or RNAs by size if not by the sequence of the bases they contain Nucleic Acid Hybridization Big idea Remember that DNA can undergo base pairing with a complementary strand of DNA or with a complementary strand of RNA this process is essential for replication and transcription However largely complementary sequences can do this too Furthermore this process is reversible heating up a solution of nucleic in the presence of low salt tends to cause complementary sequences to fall apart melt whereas lower temperatures and higher salt solutions allow them to stick together hybridize or anneal Even if the nucleic acid we want to detect is immobilized in a fixed cell or on a membrane we can still make use of this key property In that case the sequence we use to hybridize to the membrane or tissue is called a probe see Purves et al 4e Fig 13 1 DNA Cloning Big idea We want to get DNA in a form that is useful for making lots of it To do this we chop the DNA with a restriction enzyme producing sticky ends and then we ligate the sticky DNA into a vector a piece of bacterial or yeast DNA that has been similarly rended sticky Then we let bacteria do all the work of making this new DNA for us In a variation of this technique we first take mRNA and make complementary or cDNA by using an enzyme called reverse transcriptase Then we make this single stranded DNA double stranded and then we clone this DNA See Campbell 5e Fig 20 2 DNA Libraries Big idea We need a way to study genes outside the animal after all genes are pretty small One way to do this is to make a library of DNA by taking a big mixture of DNA chopping it up and putting it into a vector DNA from something like a bacterium or a bacterial virus called a phage that allows us to make lots of different kinds of DNA that can be further studied Once the libraries are made they can be screened using various methods to pull out pieces of DNA that are interesting Library construction is no longer common in labs that work with standard model organisms see Campbell 5e Fig 20 6 Kinds of DNA Big idea There are two types 1 Genomic DNA made from the DNA in chromosomes Genomic DNA will have all of the regulatory elements promoter sites etc and introns 2 cDNA is made from reverse transcribed RNA representing in the correct proportions the mRNA that was being made by the cell of interest To make cDNA we first take mRNA and make complementary or cDNA by using an enzyme called reverse transcriptase Then we make this single stranded DNA double stranded and then we can clone this DNA cDNA reflects mRNA so does not contain intronic sequences Genomic DNA regulatory and intronic sequences Enhancer distal control elements DNA Poly5A signal sequence Proximal control elements Exon Upstream Intron Promoter Primary RNA transcript 5 Exon Intron Downstream Transcrip on Exon Intron Exon Intron Exon RNA processing Intron RNA mRNA no intronic sequences Termina on region Exon Cleaved 3 end of primary