Genetics fundamentals and DNA toolkit. Partha Roy

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1 Genetics fundamentals and DNA toolkit Partha Roy 1

2 (REVIEW of Terminologies and Concepts) Haploid organism: single copy of chromosome (ex: bacteria, yeast) Diploid organism: two copies of chromosome (paternal and maternal) ex: fruit-fly(drosophila), mouse, human a pair of homologous chromosome 2n 4n p 2n p m p p m m p- paternal m- maternal Somatic cells (body cells) interphase Homologous chromosome MITOSIS p m 2n 2

3 haploid: gametes (sperm or egg) have single copy of chromosome (source : Germ cells) p m 2n Premeiotic GERM CELL 2n p p p pmm 4n Interphase ep 2n 1st Division m 2 nd Division Gametes p p m m n n n n MEIOSIS 3

4 Genetic Recombination (crossing-over) occurs between homologous chromosomes in Meiosis p 2n p p 4n m m Interphase Premeiotic GERM CELL Homologous Recombination End-products (gametes): p p m m Exchange of genetic info p p/m p/m m p p/m p/m m n n n n Increases genetic diversity: actual possible of recombination is more 4

5 information flow pathway DNA (gene) mrna Protein (molecular machinery) transcription translation Transcription + Translation = Gene Expression 5

6 Alteration in DNA sequence (mutation) Altered protein Types of mutation 1) Deletion of whole or part of a gene: Absence or truncated version of protein 2) Point mutation (a single base-pair change in DNA sequence): Examples: substitution by a different amino acid Wild-type (normal or common) GCA GAC GAA CAC GCA GGC GAA CAC Mutation: D51G A D E H A G E H (Missense Mutation) Position # GCA GAC GAA CAC A D E H GCA GAC TAA CAC A D Stop Nonsense Mutation Position # GCA GAT GAA CAC No effect A D E H 6

7 Types of mutation (contd.) 3) Insertional mutation (addition of base pairs): frame-shift (if n is not a multiple of 3) altered, longer or shorter protein sequence Examples: Wild-type mutation mutation GCA GAC GAA CAC ATT AAC TAA A D E H I N Stop GCA GAC GAA TCA CAT TAA C A D E S H Stop GCA GAC GAA TTC ACA TTA AC A D E F T L Reading frame shift (n=1) Reading Frame shift (n=2) Cont. (until it hits a stop codon) mutation GCA GAC GAA TGC CAC ATT AAC TAA A D E C H I N Stop NO Frame shift (n=3) 7

8 Examples of diseases with genetic-links Hemophilia (complete loss or altered form of factor VIII): bleeding disorder Duchenne muscular dystrophy y (altered form of dystrophin) muscle disorder Cystic fibrosis (altered form of cystic fibrosis transmembrane conductance regulator (CFTR)) pulmonary disorders d 8

9 Allele: alternative forms of gene (e.g. wild-type [WT] vs mutant paternal vs maternal) Possible alleles Haploid organism: Either WT or mutant Diploid organism: Either WT/WT or mutant/mutant t/ t tor WT/mutant t Homozygous Heterozygous Genotye Gamete WT/WT WT mut/mut mut WT/mut WT, mut 9

10 Is mutation always associated with changes in behavior or appearance (phenotype)? Assumption: No gene redundancy or compensation, mutation capable of inducing phenotype Haploid: yes; can be lethal (essential gene) Diploid: ilid may or may not be Homozygous (mut/mut): phenotype Heterozygous (WT/mut): phenotype (DOMINANT mutation) NO phenotype (RECESSIVE mutation) WT mut WT mut DOMINANT mutation (mut wins over WT) * Note : in haploids, all mutations are DOMINANT RECESSIVE mutation (WT wins over mut) Phenotype only if homozygous 10

11 Effect of mutation on gene function Mutation Or activity of gene product Haploid: Or activity of gene product Diploid: 1) Recessive : activity of gene product (loss-of-function) (otherwise one would have seen phenotype associated with overall increased activity it in heterozygous mutants) t 2) Dominant: can be either gain-of or loss-of function Dominant negative mutation 11

12 Dominant and Recessive mutations can be distinguished by the pattern of inheritance Normal male (A/A) X Mutant female (A*/A*) sperms (A) (A*) eggs Generation F1 (A/A*) mutant Generation F2 (F1xF1): gametes A or A* A A* A AA AA* mutant WT Mutant : WT =3:1 A* AA* mutant A*A* mutant Inheritance of Dominant mutation 12

13 Dominant and Recessive mutations can be distinguished by the pattern of inheritance Normal male (A/A) X Mutant female (A*/A*) sperms (A) (A*) eggs Generation F1 (A/A*) WT Generation F2 (F1xF1): gametes A or A* A A* A AA AA* WT WT Mutant : WT =1:3 A* AA* WT A*A* mutant Inheritance of Recessive mutation 13

14 Four basic tasks: 1) Separate a mixture of DNAs gel electrophoresis 2) Detect your DNA of interest Southern blot 3) Manipulate/ Engineer DNA ( Cut and Paste restriction enzyme, ligation) 3) Selectively isolate your DNA of interest from a mixture 14

15 DNA Technique #3: Selectively isolate your DNA of interest from a mixture by PCR (polymerase chain reaction)- Kary Mullis - amplifies DNA from a selected region by billionfold Region of interest What you HAVE (many copies) What you WANT Principle: DNA replication (template DNA,,p primer, dntp [A, G, T, C], DNA polymerase) Step 1: Construct two primers (A and B) Strand 1 flanking the region of interest Strand 2 A B (complementary) (primer A same as initial portion of strand 1 primer B same as initial portion of strand 2) Prepare the rxn mixture (DNA mix, two primers, dntp (A, G, T, C), thermostable DNA polymerase (Taq ) 15

16 Cycle 1 Step 1: Initial i separation of strands by heating (occurs at ~95 degree C) Step 2: Hybridization of primers at respective ends (annealing ~ degree C) A B Strand 1 Strand 2 Step 3: Extension by DNA polymerase using primers A and B(~ degree C) Strand 1 Strand 2 Separation + Annealing + Extension : 1 cycle 1 st cycle : nothing interesting (two longer strands) 16

17 Cycle 2 A B A B Separation + Annealing Extension 2 correct strands 4 longer strands # correct ds DNA=0 Cycle 3 Separation + Annealing Extension 8 correct strands, 6 longer strands # correct ds DNA=2 17

18 After that exponential growth of # correct ds products # of cycle # of correct ds products ,073,741,824 18

19 Sidenote: Application of PCR in forensics (DNA fingerprinting) Principle: Human DNA contains many microsatellite DNA island that have tandem repeats (unique to different individuals). exon1 intron1 exon2 intron2 exon3 Example: GCTGGATGGTGA. Tandem repeats Individual 1 Individual 2 Individual 3 Homologous chromosome Variable number tandem repeat (VNTR) 19

20 DNA fingerprinting (cont) Individual 1 Individual 2 Individual 3 PCR PCR products Gel electrophoresis Unique DNA fingerprinting Individual # Ex: Comparison of DNA fingerprinting of a potential suspect with the same from the DNA sample obtained at the crime scene 20