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

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. DNA mRNA protein • 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 • DNA mRNA • Direct positional cloning • DNA • All three options require the cloning of DNA.

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 cutter 44 = 256 bp • 5-base cutter 45 = 1,024 bp • 6-base cutter 46 = 4,096 bp • 8-base cutter 48 = 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 EcoRI 5’…GAATTC…3’ 5’…G AATTC…3’ 3’…CTTAAG…5’ 3’…CTTAA G…5’ PstI 5’…CTGCAG…3’ 5’…CTGCA G…3’ Providencia stuartii 3’…GACGTC…5’ 3’…G ACGTC…5’ BLUNT ENDS Haemophilus aegyptius HaeIII 5’…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. ori • 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 gene for tetracycline resistance gene for ampicillin resistance EcoRI Pst I Sal I

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

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 Bam HI sites “left arm” “right arm” internal fragment (dispensable for phage growth)

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

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

14. Partial restriction enzyme digestion allows cloning of overlapping fragments a “contig” • 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 109 bp) • assuming ~20 kb fragments • theoretical minimum = ~150,000 • 99% probability that every sequence is represented = ~800,000

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

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

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

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

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