1. UNIT 2 MANIPULATION OF DNA AND GENE ISOLATION LECTURES: 9. DNA Cloning and Library Construction 10. Isolating Genes

2. 9. DNA Cloning and Library Construction a). DNA cloning i). Restriction endonucleases ii). Cloning vectors iii). The process of cloning a segment of DNA b). Library construction i). Genomic libraries ii). cDNA libraries

3. How does one isolate a gene for an inherited disorder? There are three options: Start with a candidate protein DNA protein Start with a candidate mRNA DNAmRNA Direct positional cloning DNA All three options require the cloning of DNA. DNA mRNA protein

4. Restriction endonucleases Restriction enzymes cut DNA into specific fragments Restriction enzymes recognize specific base sequences in double-stranded DNA and cleave both strands of the duplex at specific places Characteristics of restriction enzymes: 1. Cut DNA sequence-specifically 2. Bacterial enzymes; hundreds are purified and available commercially 3. Restriction-modification system Bacteria have enzymes that will cleave foreign DNA; hence, “restrict” the entry of viral DNA. To prevent the bacteria’s own DNA from being cut, there is a second enzyme that methylates the same sites recognized by the restriction enzyme (modifies that site). 4. Named (e.g., EcoRI) for bacterial genus, species, strain, and type 5. Recognize specific 4-8 bp sequences sequences have symmetry (they are palindromes) after cutting the DNA, the cut ends are either blunt staggered (overhangs) - cohesive ends facilitate cloning the DNA 6. Frequency of cutting 4-base cutter4 4 = 256 bp 5-base cutter4 5 = 1,024 bp 6-base cutter4 6 = 4,096 bp 8-base cutter4 8 = 65,536 bp

5. 4-base cutter: cuts DNA into 256 bp average-sized fragments in a random sequence every 256 bp: NO 256 bp average-size fragments: YES Bar = 256 bp

6. Products generated by restriction enzymes COHESIVE ENDS EcoRI5’…GAATTC…3’5’…GAATTC…3’ 3’…CTTAAG…5’3’…CTTAA G…5’ PstI5’…CTGCAG…3’5’…CTGCA G…3’ Providencia stuartii 3’…GACGTC…5’3’…GACGTC…5’ BLUNT ENDS Haemophilus aegyptius HaeIII5’…GGCC…3’ 5’…GG CC…3’ 3’…CCGG…5’ 3’…CC GG…5’

7. Formation of recombinant DNA molecules cut DNAs mix together fragments and anneal cohesive ends seal 3’, 5’ ends by DNA ligase recombinant DNAs

8. Vectors used in molecular cloning Vector Insert (and host) Characteristics size range Plasmid Small circular DNA <5 - 10 kb (bacteria, yeast) Bacteriophage lambda Linear viral DNA up to ~20 kb or phage lambda (bacteria) Cosmid Hybrid of plasmid up to ~50 kb (bacteria) and phage Yeast artificial DNA containing yeast~200 to ~1000 kb chromosome or YAC centromere, telomeres, (yeast) and origins of replication

9. Structure of pBR322 - a common cloning vector derived from a naturally occurring plasmid has antibiotic resistance genes for selection of transformants containing the plasmid has unique restriction enzyme cleavage sites for insertion of foreign DNA has origin of DNA replication (ori) for propagation in E. coli EcoRI Sal I gene for ampicillin resistance gene for tetracycline resistance Pst I ori

10. Cloning a segment of DNA into a plasmid vector bacteria are “transformed” with the recombinant plasmid colonies that grow in tetracycline, but not in ampicillin are isolated PstI Human DNA cut with PstI P P pBR322 amp R, tet R pBR322 (human clone) tet R P P amp R tet R pBR322 DNA cut with PstI inactivating the amp R gene tet R combine and ligate

11. Library construction two types of libraries a genomic library contains fragments of genomic DNA (genes) a cDNA library contains DNA copies of cellular mRNAs both types are usually cloned in bacteriophage vectors Construction of a genomic library vector DNA (bacteriophage lambda) lambda has a linear double- stranded DNA genome the left and right arms are essential for the phage replication cycle the internal fragment is dispensable “left arm”“right arm” Bam HI sites internal fragment (dispensable for phage growth)

12. NNG GATCCNN NNCCTAG GNN internal fragment cut with Bam HI (6-base cutter) remove internal fragment “left arm”“right arm” cut with Sau 3A (4-base cutter) which has ends compatible with Bam HI: NNN GATCNNN NNNCTAG NNN isolate ~20 kb fragments human genomic DNA (isolated from many cells) Bam HI sites:

13. combine and treat with DNA ligase “left arm”“right arm” “left arm”“right arm” package into bacteriophage and infect E. coli 1 2 3 4 5 6 genomic library of human DNA fragments in which each phage contains a different human DNA sequence 7

14. isolation of ~20 kb fragments provides optimally sized DNAs for cloning in bacteriophage partial digestion with a frequent-cutter (4-base cutter) allows production of overlapping fragments, since not every site is cut overlapping fragments insures that all sequences in the genome are cloned overlapping fragments allows larger physical maps to be constructed as contiguous chromosomal regions (contigs) are put together from the sequence data number of clones needed to fully represent the human genome (3 X 10 9 bp) assuming ~20 kb fragments theoretical minimum = ~150,000 99% probability that every sequence is represented = ~800,000 Partial restriction enzyme digestion allows cloning of overlapping fragments a “contig”

15. All possible sites: Results of a partial digestion: = uncut= cut

16. Construction of a cDNA library reverse transcriptase makes a DNA copy of an RNA The life cycle of a retrovirus depends on reverse transcriptase retrovirus 1. virus enters cell and looses envelope 2. the capsid is uncoated, releasing genomic RNA and reverse transcriptase 3. reverse transcriptase makes a DNA copy 4. then copies the DNA strand to make it double-stranded DNA, removing the RNA with RNase H 5. the DNA is then integrated into the host cell genome where it is transcribed by host RNA polymerase II 6. it is translated into viral proteins, and assembled into new virus particles new viruses

17. cDNA library construction AAAAA 5’3’ mRNA (all mRNAs in cell) anneal oligo(dT) primers of 12-18 bases in length AAAAA TTTTT 5’3’ 5’ add reverse transcriptase and dNTPs AAAAA TTTTT 5’ 3’ 5’ cDNA add RNaseH (specific for the RNA strand of an RNA-DNA hybrid) and carry out a partial digestion AA TTTTT 5’ short RNA fragments serve as primers for second strand synthesis using DNA polymerase I 3’

18. AAAAA TTTTT 5’ 3’ short RNA fragments serve as primers for second strand synthesis using DNA polymerase I AAA TTTTT 5’ 3’ DNA polymerase I removes the remaining RNA with its 5’ to 3’ exonuclease activity and continues synthesis DNA ligase seals the gaps AAA TTTTT 5’ 3’ AAAAA TTTTT 5’ 3’ double-stranded cDNA

19. AAAAA TTTTT 5’ 3’ NNNNNNNNG NNNNNNNNCTTAA EcoRI linkers are ligated to both ends using DNA ligase AAAAANNNNNNNNG TTTTTNNNNNNNNCTTAA double-stranded cDNA copies of mRNA with EcoRI cohesive ends are now ready to ligate into a bacteriophage lambda vector cut with EcoRI 5’ 3’ AATTCNNNNNNNN GNNNNNNNN

20. EcoRI sites combine cDNAs with lambda arms and treat with DNA ligase “left arm”“right arm” “left arm”“right arm” package into bacteriophage and infect E. coli 1 2 3 4 5 6 cDNA library in which each phage contains a different human cDNA 7 cDNAs