Chapter 2

DNA Biology Review

©2002 Academic Press

Target Region for PCRTarget Region for PCR

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotides

DNA in the Cell

©2002 Academic Press

Basic Components of Nucleic Acids

O

Base(A, T, C, or G)

HO

1’

3’ 2’

4’

5’CH2OP

H

HH

H

O

O-

HO

O

Base(A, T, C, or G)

HOH

1’

3’ 2’

4’

5’CH2OP

H

HH

H

O-

HO

5’end|

Phosphate|

Sugar—Base…|

Phosphate|

Sugar—Base…|

3’end

PhosphatePhosphate

SugarSugar

BaseBase

©2002 Academic Press

Base Pairing of DNA Strands

A = TG C

T = A

A = T

C G

T

CCA

GG

TA

G C

T = A

T = A

C G

A = T

A = TG C

5’

3’

5’

3’

5’ 3’

3’ 5’denatured

strands

hybridizedstrands

Hydrogen bonds

C G C

G

G C

Phosphate-sugar backbone

©2002 Academic Press

Basic Chromosome Structure

p(short arm)

centromere

telomere

q(long arm)

telomere

Band 5

Band 3

Chromosome 12

12p312p3

12q512q5

©2002 Academic Press

htt

p:/

/ww

w.n

cbi.n

lm.n

ih.g

ov/

ge

no

me

/gu

ide

/

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

Human Genome Contains 23 Pairs of Chromosomes

Sex-chromosomes

©2002 Academic Press

TYPES OF DNA MARKERS

• Length Variation– short tandem repeats (STRs)

• microsatellites• simple sequence repeats (SSRs)

– minisatellites (VNTRs)

• Sequence Variation – single nucleotide polymorphisms (SNPs)– insertions/deletions

(GATA)(GATA)(GATA)(GATA)

(G/A)

(CA)(CA)(CA)(CA(CA)(CA)

©2002 Academic Press

Family Inheritance of STR Alleles (D13S317)

Father

Child #1

Child #2

Child #3

Mother

PCR product size (bp)

11 14

11

12 14

8 14

12

128

©2002 AP, Elsevier

Characteristic RFLP Methods PCR Methods

Time required to obtain results

6-8 weeks with radioactive probes; ~1 week with chemiluminescent probes

1-2 days

Amount of DNA needed 50-500 ng 0.1-1 ng

Condition of DNA needed high molecular weight, intact DNA

may be highly degraded

Capable of handling sample mixtures

Yes (single locus probes)

Yes

Allele identification Binning required Discrete alleles obtained

Power of Discrimination ~1 in 1 billion with 6 loci ~1 in 1 billion with 8-13 loci (requires more loci)

Comparison of RFLP and PCR ©2002 AP, Elsevier