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Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III I II III I II III Radioactively labeled probe for CD4 gene RNA macrophage B cells T cells Film over blot Probe detection Hybridization with radioactive probe Probe base-pairs with mRNA Nitrocellulose blot 1 4 5 3 2 Northern Blot (to detect CD4 mRNA)

Fig. 20-11

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Northern Blot (to detect CD4 mRNA). TECHNIQUE. Heavy weight. I II III. RNA. Nitrocellulose membrane (blot). Gel. Sponge. T cells. B cells. macrophage. Paper towels. Fig. 20-11. Alkaline solution. 2. 1. 3. Preparation of restriction fragments. DNA transfer (blotting). - PowerPoint PPT Presentation

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Page 1: Fig. 20-11

Fig. 20-11TECHNIQUE

Nitrocellulosemembrane (blot)

Alkalinesolution

DNA transfer (blotting)

Sponge

Gel

Heavyweight

Papertowels

Preparation of restriction fragments Gel electrophoresis

I II III

I II IIII II III

Radioactively labeledprobe for CD4 gene

RNA

macrophageB cellsT cells

Film overblot

Probe detectionHybridization with radioactive probe

Probe base-pairswith mRNA

Nitrocellulose blot

1

4 5

32

Northern Blot (to detect CD4 mRNA)

Page 2: Fig. 20-11

Fig. 20-13

TECHNIQUE

RESULTS

Gel electrophoresis

cDNAs

CD4 mRNA

PCR amplification

Different cell types

Primers

1 2 3 4 5 6

mRNAscDNA synthesis 1

2

3

Reverse Transcriptase PCR (RT-PCR) to detect CD4 mRNA

Page 3: Fig. 20-11

Fig. 20-15TECHNIQUE

Isolate mRNA.

Make cDNA by reversetranscription, usingfluorescently labelednucleotides.

Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.

Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA fragmentsrepresentingspecific genes

DNA microarraywith 2,400human genes

DNA microarray

1

2

3

4

Microarrays to detect many (or all) mRNAs at once

Page 4: Fig. 20-11

WT

dif1

∆ dif1

myb98

∆ myb98

genes

Example of array data

Page 5: Fig. 20-11

Table 21-1

Page 6: Fig. 20-11

Human Genome Project (Multinational Consortium)1990-2003

Entire 3 x 10^9 nucleotide sequence of a composite haploid human genome

~$500 million - $1 billion

Celera Genomics (Private Company)1998-2003

Shotgun sequencing approach

~ $300 million

“Divide and conquer” approach

Page 7: Fig. 20-11
Page 8: Fig. 20-11
Page 9: Fig. 20-11

Fig. 20-12a

DNA(template strand)

TECHNIQUE

DNA polymerase

Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

dATP

dCTP

dTTP

dGTP

ddATP

ddCTP

ddTTP

ddGTP

How can we sequence DNA? (Sanger dideoxy method)

Page 10: Fig. 20-11

Fig. 20-12bTECHNIQUE

RESULTS

DNA (template strand)

Shortest

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Directionof movementof strands

Detector

Last baseof longest

labeledstrand

Last baseof shortest

labeledstrand

Page 11: Fig. 20-11

Linkage mapping1

2

3

Geneticmarkers

Physical mapping

Overlappingfragments

DNA sequencing

How can we sequence an entire genome?

Page 12: Fig. 20-11

Linkage mapping1

2

3

Geneticmarkers

Physical mapping

Overlappingfragments

DNA sequencing

How can we sequence an entire genome?

Genome sequencing:Divide and conquer approach

-Ordered, large fragments of chromosomes are cloned

Page 13: Fig. 20-11

Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carryingplasmids

RESULTS

Colony carrying non-recombinant plasmidwith intact lacZ gene

One of manybacterialclones

Colony carrying recombinant plasmid with disrupted lacZ gene

Page 14: Fig. 20-11

Linkage mapping1

2

3

Geneticmarkers

Physical mapping

Overlappingfragments

DNA sequencing

Genome sequencing:Divide and conquer approach

- Ordered, large fragments of chromosomes are cloned

-Each fragment is sequenced

How can we sequence an entire genome?

Page 15: Fig. 20-11

Cut the DNAinto overlappingfragments short enoughfor sequencing

1

2

3

4

Clone the fragmentsin plasmid or phagevectors.

Sequence eachfragment.

Order thesequences intoone overallsequencewith computer software.

“Shotgun” sequencing approach

Page 16: Fig. 20-11

TTATTTCCCATTTTTCTTAAAAAGGAAGAACAAACTGTGCCCTAGGGTTTACTGTGTCAGAACAGAGTGTGCCGATTGTGGTCAGGACTCCATAGCATTTCACCATTGAGTTATTTCCGCCCCCTTACGTGTCTCTCTTCAGCGGTCTATTATCTCCAAGAGGGCATAAAACACTGAGTAAACAGCTCTTTTATATGTGTTTCCTGGATGAGCCTTCTTTTAATTAATTTTGTTAAGGGATTTCCTCTAGGGCCACTGCACGTCATGGGGAGTCACCCCCAGACACTCCCAATTGGCCCCTTGTCACCCAGGGGCACATTTCAGCTATTTGTAAAACCTGAAATCACTAGAAAGGAATGTCTAGTGACTTGTGGGGGCCAAGGCCCTTGTTATGGGGATGAAGGCTCTTAGGTGGTAGCCCTCCAAGAGAATAGATGGTGAATGTCTCTTTTCAGACATTAAAGGTGTCAGACTCTCAGTTAATCTCTCCTAGATCCAGGAAAGGCCTAGAAAAGGAAGGCCTGACTGCATTAATGGAGATTCTCTCCATGTGCAAAATTTCCTCCACAAAAGAAATCCTTGCAGGGCCATTTTAATGTGTTGGCCCTGTGACAGCCATTTCAAAATATGTCAAAAAATATATTTTGGAGTAAAATACTTTCATTTTCCTTCAGAGTCTGCTGTCGTATGATGCCATACCAGAGTCAGGTTGGAAAGTAAGCCACATTATACAGCGTTAACCTAAAAAAACAAAAAACTGTCTAACAAGATTTTATGGTTTATAGAGCATGATTCCCCGGACACATTAGATAGAAATCTGGGCAAGAGAAGAAAAAAAGGTCAGAGTTTAATCCTCATTCCTAAGTTATGTAAACCAAAAATAAAATTCTGAAGATGTCCTGATCATCTGAATGGACCCTTCCTCTGGACCAGGGCATTCCAAAGTTAACCTGAAAATTGGTTTGGGCCATGATGGGAAGGGAGGTTTGGATATGCCTCATTATGCCCTCTTCCCTTTCAGAATTCAGGAAAAGCCAACCAGCATTAACATCAACACAGATTTTCAGATCTTAGGTTTCTTTCCGATCTATTCTCTCTGAACCCTGCTACCTGGAGGCTTCATCTGCATAATAAAACTTTAGTCTCCACAACCCCTTATCTTACCCCAGACATTCCTTTCTATTGATAATAACTCTTTCAACCAATTGCCAATCAGGGTATGTTTAAATCTACCTATGACCTGGAAGCCCCCACTTTGCACCCTGAGATCAAACCAGTGCAAATCTTATATGTATTGATTTGTCAATGAAAACAGTCAAAGCCAGTCAGGCACAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTAGATCACCTGAGGTCAGGAGTTCGACACCAGCCTGGCCAACATGGTGAAACCCCGTCCCTACTAAAATACAAAAATTAGCCCAGCTTGGTGGTGGGCACCTGTAATCTTAGCTACTGCAGAGACTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAGTGACCTGAGATTTTGCCATTGCACTCCAGCCTGGGCAACAGAGCAAGACTCTATCTCAAAAAACAAACAAACAAACAAACAAACAAACAAACTGTCAAAATCTGTACAGTATGTGAAGAGATTTGTTCTGAACCAAATATGAATGACCATGGTCCATGACACAGCCCTCAGAAGACCCTGAGAACATGTGCCCAAGGTGGTCACAGTGCATCTTAGTTTTGTACATTTTAGGGAGATATGAGACTTCAGTCAAATACATTTTTAAAAAATACATTGGTTTTGTCCAGAAAGCCAGAACCACTCAAAGCAGGGGTTTCCAGGTTATAAGTAGATTTAAAATTTTTCTGATTGACAATTGGTTGAAAGAGTTGTCAATAGAAAGGAATGTCTGCATTGTGACAAGAGGTTGTGGAGACCAAGTTTCTGTCATGCAGATGAAGCCTTCAGGTAGCAGGCTTCCAAGATAACAGGTTGTAAATAGTTCTTATCAGACTTAAGTTCTGTGGAGACGTAAAATGAGGCATATCTGACCTCCACTTCCAAAAACATCTGAGACAGGTCTCAGTTAATTAAGAAAGTTTGTTCTGCCTAGTTTAAGGACATGCCCATGACACTGCCTCAGGAGGTCCTGACAGCATGTGCCCAAGGTGGTCAGGATACAGCTTGCTTCTATATATTTTAGGGAGAAAATACATCAGCCTGTAAACAAAAAATTAAATTCTAAGGTCCCTGAACCATCTGAATGGGCTTTCTTCTAGGCCAGGGCACTCTAAAATTGAAGAACCTGAACATTCCTTTCTATTGATAATACTTTCAGCCAGTTGAGCCCATTCAGACCACAGCAAGGTGCCAGGCCAGGCAAGGGCTGACTTGAGATACCTGCCAGATGAGTCACTGGCAAAAGGTGCTGCTCCCTGGTGAGGGAGAAACACCAGGGGCTGGGAGAGGCCCAGAAGGCTCTGAAGGAGTTTTGGTTTGGCTGGCCATGTGTGCAATTAGCGTGATGAGCTCTGACATGGCCTTGCATGGACGGATTGGGCAGGACACCCCAGCTGAGGAGGATGGCAGGAGTGATGGCACAGGGGAAAGGGTGGCATACCCAGGTGACAGCTCCCCACTACCTCCACTCTGTGCTGCAGCTCAGGGGCTGGGTCTTCTGCTGCAACTCAGCCCCTCTGTACCAGCCCTGGCCTCATTCCCTTGGTTCCAGGACACCCAGCTGACAAAAGGGACTTGCCTGTACCCCTGCACCTGGTCCTACACCTGGCTCCTGGGTTGTCAGCAGGTGTTTGTTGGGCCAACGAGTGCATGGATGGAAACACAGACAGAAGGACAGATGGAGAGATGGTGGGTGGCCAGACAAAGGAGTAACTTGGTGAGGAATGTGCATTAGGAAATCACAGAAGAGCAGAAACTGTTTGAAAATTCCAAGTGGGGAAAGTGAGGAGGTGAAGCAGGGCTGAAGGGCCTCCCTCAGAGCCTTCTCCCACTCTGTGGTGTCCACATCCCCTTGGTCGTCCTTGTGGGAGGCACTCACCTTTTGCTCAGCCTATTGTGGCTACAGCCCAGCAGGTCCCAGGTGGCACCAGCCAAGATGAAGGTGGCATTGAGGGCTGAAGTCTCCCTCACCATGAAGGGATGATGTATAGTGGGTGGGGCCTCAGGAGGAAGAGGGCCACCAACCCTACCTGGCCCCTAACCTGCTGCCTGGAGTAGGCAGGTACCAGAGGCATGGGGTGAGGCATGTTGCAGGTCGAGGACCAGGGCCATCTCACTGCCTGAGCCCATGGACTGGCTCAGGGGTCTGTCAGATGATTCTAGAGCTGAGTTGGAGGTAAGGGCAGGGGGTTTGTTCCTGGGTTCAAGACCATGGAAGGAAGGGGTAGAGAAGGAGGCCAACAAGTGAGGAGGCAAATTACAGTGGCTGGCAGAAGGAGAGAGAAGCCAGGACAGGTGGCTGTGGCCCTGTCCCTGCAGGCAGACCCAGGAAGGAGCTCAGAGACAGGATTCATGCCAAGCCTGCCTACCCAGCACATCTCTCCTCATGGACATGAGAGAAAACCCTCCAGCTTGGCCCTCACATCTGTGAAACCCACAGTAATGGGGCTGACATCCTCTGCCCTATGCAAGAGAGGTTTCCCAAGCACTTGCAGCAAGTGAGACTGCACAGGATGGCGAATCCACAAAGAACACGTTGTTCTCATGCTCTTTGGAAGCACCAATTTACATTCTG

The Human Genome (partial sequence….)

Page 17: Fig. 20-11

Table 21-1

Page 18: Fig. 20-11

Fig. 21-7Exons (regions of genes coding for protein

or giving rise to rRNA or tRNA) (1.5%)

RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)

Introns andregulatorysequences(24%)

UniquenoncodingDNA (15%)

RepetitiveDNAunrelated totransposableelements (15%)

L1sequences(17%)

Alu elements(10%)

Simple sequenceDNA (3%)

Large-segmentduplications (5–6%)

Page 19: Fig. 20-11

Comparison of gene organization in different species (centered on region containing RNA polymerase gene)

Page 20: Fig. 20-11

Fig. 21-9a

TransposonNew copy oftransposon

DNA ofgenome Transposon

is copiedInsertion

Mobile transposon

(a) Transposon movement (“copy-and-paste” mechanism)

Page 21: Fig. 20-11

Fig. 21-9b

RetrotransposonNew copy of

retrotransposon

Reversetranscriptase

Insertion

RNA

(b) Retrotransposon movement

Page 22: Fig. 20-11

TECHNIQUE

Gel electrophoresis

cDNAs

PCR amplificationPrimers

mRNAscDNA synthesis 1

2

3

Reverse Transcriptase PCR (RT-PCR)

Large scale sequencing of cDNA fragments

Sequence large numbers (millions) of cDNA fragments

Page 23: Fig. 20-11

No UV(3 samples)

UV(3 samples)

Large scale sequencing of cDNA fragments

Fragments matching rad51


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