Reading Lecture 8: Lecture 9: Lecture 8. DNA Libraries. Definition Types Construction

Transcription

1 Lecture 8 Reading Lecture 8: Lecture 9: DNA Libraries Definition Types Construction 142

2 DNA Libraries A DNA library is a collection of clones of genomic fragments or cdnas from a certain organism or tissue Fragments of DNA representing the entire genome in a series of vectors Want the libraries to represent ALL the genes or transcripts of an organism 143

3 Reasons to Make a Library To identify and isolate a gene of interest Source of material To sequence a genome Need every piece of DNA so can find order of bp To map a genome Identify gene location on a chromosome To study/isolate expressed genes under different conditions cdna library 144

4 145 Cloning and identifying genes

5 Genomic Libraries Collections of clones with inserts corresponding to the genomic DNA of an organism ALL the DNA Includes exons, introns, promoters, junk DNA Want complete representation of genome Fragments of similar size Random distribution Overlapping 146

6 Making a library with clones representing the entire genome Cut genomic DNA with restriction enzymes Impossible with complete digest with single restriction enzyme Fragments vary in size and do not overlap B B B B B 147

7 Making a library with clones representing the entire genome Cut genomic DNA with restriction enzymes Partial digest with 4 bp cutter Ligate into vector BamHI sites Sau3A sites Fragments from partial Sau3A digest 148 Random shearing Add adaptors or blunt end ligation Makes libraries with uniform size inserts

8 Clones from libraries with overlapping fragments can be placed in order to form contigs (contiguous sequences) A B C D E

9 Number of Clones Needed Can calculate the number of clones needed to obtain a representative library N = ln (1-p) ln (1-f) N = number of clones f = fraction of genome in clone p = probability 150

10 Number of Clones Needed Depends on Genome complexity Insert size Desired probability of inclusion Approximate # of clones required for Organism complete library (20 kb inserts, 99% probability) E. coli 1,500 Yeast 4,600 Mammals 650,

11 Vectors for making genomic libraries Desirable features: Efficiency of cloning Capacity for large inserts Stability in host Maximum DNA insert possible in various vectors 152

12 An early strategy for producing a representative genomic library from a complex eukaryote (Fig. 6.2) One scheme for making a library in a cosmid clone (Fig. 5.3) 153

13 Strategy to insert large DNA fragments into a BAC (up to 300 kb) Pulse field gel electrophoresis 154

14 cdna Libraries Collection of clones with inserts corresponding to expressed genes Complementary DNA (cdna) DNA copies of mrna Represents expressed genes No introns, promoters, junk DNA Made from 155 Specific tissues Certain conditions Different stages of development Useful for: Comparing expression patterns Splice variants Identifying genes

15 Making cdna Libraries Isolate total RNA Phenol-chloroform extraction Isolate mrna Basis of separation PolyA tail of Eukaryotic mrna Oligo dt 156

16 cdna Synthesis Reverse Transcriptase Generation of double-stranded DNA from single-stranded mrna Uses enzyme reverse transcriptase Purified from a retrovirus Virus that transmits itself as RNA then converts to DNA once in host cell by the enzyme reverse transcriptase Reverse transcribes DNA off of a RNA strand Like DNA polymerase Needs a 3 OH 157

17 cdna Synthesis Step 1. First strand synthesis Oligo dt primer Reverse transcriptase Step 2. RNaseH Cleaves RNA portion of RNA:DNA hybrid Step 3. DNA polymerase Uses first strand as template to make second strand 158

18 cdna Synthesis- Alternatives Oligo dt Random hexamers Better distribution Gene-specific primers More selective 159

19 Average length of a cdna Average protein is 44,000 Da 1 aa = 110 Da Average protein has 400 aa 3 bp/aa Average ORF is 1200 bp 3 UTR and polya Add 300 bp more Average cdna is 1500 bp long 160

20 Inserting cdnas into Vectors Blunt end cloning Use adaptors Short synthetic duplex oligonucleotide Contains restriction enzyme site Attach to ends of cdna Open vector with same enzyme Ligate compatible ends 161

21 Challenges with cdna Libraries Transcript abundance 162 Differential splicing Obtaining full length cdna clones

22 Rapid Amplification of cdna Ends (RACE) To obtain a full-length cdna from a partial clone Reverse transcriptase with gene-specific primer Attach new end (AAAA) with terminal transferase PCR to amplify with TTTT and gene-specific primers