Green with envy?? Jelly fish “GFP” Transformed vertebrates.

Green with envy??Jelly fish “GFP”

Transformed vertebrates

DNA Manipulation, Gene Cloning and Karyotyping

Gene Cloning• Techniques for gene cloning enable scientists

to prepare multiple identical copies of gene-sized pieces of DNA.

• Most methods for cloning pieces of DNA share certain general features.– For example, a foreign gene is inserted into a

bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell.

– Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents.

– Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.

• One goal may be to produce a protein product for use.

• A second goal may be to prepare many copies of the gene itself.– This may enable scientists to determine the

gene’s nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.

Restriction Enzymes

• In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria.

• Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.

• Restriction enzyme cleaves DNA at specific sequence of bases called a restriction site.– often a symmetrical series of four to eight bases

on both strands running in opposite directions.– restriction site on one strand is 3’-CTTAAG-5’, the

complementary strand is 5’-GAATTC-3

• Restriction enzymes cut the sugar phosphate backbone bonds of both DNA strands, creating single-stranded ends, sticky ends.– These extensions will form hydrogen-bonded

base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme

• Recombinant plasmids--produced by splicing restriction fragments from foreign DNA into plasmids.– Plasmid is a circular piece of DNA found in

bacteria and contain genes.– Plasmids can be used to insert DNA from

another organism into a bacterial cell.

• Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it.

Recombinant DNA vectors

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

• Cloning a gene into a bacterial plasmid can be divided into five steps.


Fig. 20.3

Blue colonies White colonies

• When the source of DNA is small or impure, the polymerase chain reaction (PCR) is quicker and more selective. (limitation of PCR -- produces short DNA segments within a gene and not entire genes.)

• This technique can quickly amplify any piece of DNA without using cells.

• Devised in 1985, PCR has had a major impact on biological research and technology.

The polymerase chain reaction (PCR) clones DNA entirely in vitro


• The DNA is incubated in atest tube with special DNA polymerase, a supply of nucleotides,and short pieces ofsingle-stranded DNA as a primer.


Fig. 20.7

• PCR can make billions of copies of a targeted DNA segment in a few hours.– This is faster than cloning via recombinant

bacteria.

• PCR, a three-step cycle: – heating, – cooling, – replication, – brings about a chain reaction that produces an

exponential number of DNA molecules.

• PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material

• PCR has amplified DNA from a variety of sources:– fragments of ancient DNA from a 40,000-year-

old frozen wooly mammoth,– DNA from tiny amount of blood or semen found

at the scenes of violent crimes,– DNA from single embryonic cells for rapid

prenatal diagnosis of genetic disorders,– DNA of viral genes from cells infected with

difficult-to-detect viruses such as HIV.


Human chromosome disorders • High frequency in humans

– most embryos are spontaneously aborted– alterations are too disastrous– developmental problems result from biochemical

imbalance• imbalance in regulatory molecules?

– hormones?

– transcription factors?

• Certain conditions are tolerated– upset the balance less = survivable– characteristic set of symptoms = syndrome

Chromosomal abnormalities• Incorrect numbers of

chromosomes(Numerical)– Nondisjunction

• chromosomes don’t separate properly during meiosis

• Structural abnormalities– Chromosome mutations

• Deletion

• Inversion

• Duplication

• Translocation

Numerical Abnormalities:Nondisjunction

• Problems with meiotic spindle cause errors in daughter cells– homologous chromosomes do not separate

properly during Meiosis 1– sister chromatids fail to separate during Meiosis 2– too many or too few chromosomes

2n n

n

n-1

n+1

Alteration of chromosome number

Nondisjunction • Baby has wrong chromosome number

– Trisomy • cells have 3 copies of a chromosome

– Monosomy• cells have only 1 copy of a chromosome

n+1 n n-1 n

monosomy2n-1

trisomy2n+1

Down syndrome• Trisomy 21

– 3 copies of chromosome 21– 1 in 700 children born in U.S.

• Chromosome 21 is the smallest human chromosome– but still severe effects

• Frequency of Down syndrome correlates with the age of the mother

Down syndrome & age of mother

Mother’s age

Incidence of Down Syndrome

Under 30 <1 in 1000

30 1 in 900

35 1 in 400

36 1 in 300

37 1 in 230

38 1 in 180

39 1 in 135

40 1 in 105

42 1 in 60

44 1 in 35

46 1 in 20

48 1 in 16

49 1 in 12

Rate of miscarriage due to amniocentesis: 1970s data

0.5%, or 1 in 200 pregnancies

2006 data<0.1%, or 1 in 1600 pregnancies

Sex chromosomes abnormalities

• Human development more tolerant of wrong numbers in sex chromosome

• But produces a variety of distinct syndromes in humans– XXY = Klinefelter’s syndrome male – XXX = Trisomy X female– XYY = Jacob’s syndrome male– XO = Turner syndrome female

• 2X and 1Y– one in every 2000 live births– have male sex organs, but are

sterile– feminine characteristics

• some breast development• lack of facial hair

– tall– normal intelligence

Klinefelter’s syndrome

Klinefelter’s syndrome

Jacob’s syndrome male• 1X and 2 Y

– 1 in 1000 live male births

– extra Y chromosome– slightly taller than

average– more active– normal intelligence, slight learning disabilities– delayed emotional immaturity– normal sexual development

Trisomy X• 3 X

– 1 in every 2000 live births– produces healthy females

• Why?• all but one X chromosome is inactivated

Turner syndrome• 1X

– 1 in every 5000 births– varied degree of effects – webbed neck– short stature– sterile

Changes in chromosome structure

• Deletion– loss of a chromosomal segment

• Duplication– repeat a segment

• Inversion– reverses a segment

• Translocation– move segment from one chromosome to

another

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46,XY,t(8;9)(q24.3;q22.1)

Translocation

FISH analysis: abl/bcr Genes on Diploid Cells and Ph Positive CML Cells

Normal

Translocation

Genetics Laboratory• Cytogenetics

Tissue culture

Harvesting/Slide Preparation

KaryotypingResults / Interpretation

Report

FISH

Analysis

Genetic testing• Amniocentesis in 2nd trimester

– sample of embryo cells– stain & photograph chromosomes

• Analysis of karyotype

Karyotyping

Karyotype of a normal male

Chromosome Spread

Dosage CompensationDo males have half as much of the products of

genes on the X as do females? NO!!

X Inactivation

Interphase: Chromomes can’t be stained, but a dark-staining body is visible in the nuclei of

cells of female mammals

Barr Body: Inactive X

Which X gets inactivated?Mary Lyon & Lianne Russell

(1961) proposed that one or other of X becomes inactivated at a particular time in early development. Within each cell,which X becomes inactivated is random.

As development proceeds, all cells arising by cell division after that time have same X inactivated.

In 64-cell embryos

Adult female mammals have two copies of each gene on the X chromosome.

Green with envy?? Jelly fish “GFP” Transformed vertebrates.

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