BIOCHEMISTRY & CELL BIOLOGY

SELF-INSTRUCTION MODULE 1-5

MOLECULAR GENETICS
METHODS IN MEDICINE

DEPARTMENT OF BIOCHEMISTRY
2012 by Universidad Central del Caribe
All rights reserved
PURPOSE & SCOPE OF THE SELF-INSTRUCTION MODULES

Due to the limitation of faculty-student contact hours, it is not

possible to present in the classroom all of the concepts in
Biochemistry & Cell Biology that the student must learn. The
self-instruction modules (SIMs) are designed to provide,
within a student-centered electronic format, a structured
presentation of essential concepts & information that cannot
be presented in the classroom. Each SIM is a power-point
presentation that the student may access via the Blackboard
BCB course site at his or her convenience. Studying each of
the SIMs is compulsory. The material on each SIM is tested on
the subsequent examinations.
 LEARNING OBJECTIVE OF THIS MODULE

Upon completion of this module the student should be able to

explain in detail each of the most important methods that are used
in recombinant DNA technology & molecular genetic analysis.

NOTE: THIS MODULE IS DESIGNED TO BE COMPLETED AFTER THE

STUDENT HAS LEARNED THE BASIC CONCEPTS OF NUCLEIC ACIDS
THAT ARE COVERED IN THE CORRESPONDING CONFERENCES.

NOTE: THIS MODULE IS DESIGNED TO BE COMPLETED BEFORE THE

STUDENT ATTENDS CLINICAL APPLICATION EXERCISE #2
MOLECULAR GENETICS
 CONTENTS OF THIS MODULE:

INTRODUCTION
RESTRICTION ENDONUCLEASES
RECOMBINANT DNA
SOUTHERN BLOTTING
DNA FINGERPRINTING
NORTHERN BLOTTING
BACTERIAL TRANSFORMATION
EXPRESSION OF FOREIGN GENES
POLYMERASE CHAIN REACTION (PCR)
DNA PROFILING
DNA SEQUENCING
 INTRODUCTION
DURING THE SECOND HALF OF THE 2OTH CENTURY THERE HAVE
BEEN TREMENDOUS ADVANCES IN MOLECULAR GENETICS

THESE ADVANCES HAVE HAD & CONTINUE TO HAVE MAJOR IMPACTS

ON THE UNDERSTANDING & TREATMENT OF HUMAN DISEASE

THESE ADVANCES INCLUDE:

THE DEVELOPMENT OF METHODS TO SEQUENCE DNA
THE DEVELOPMENT OF BLOTTING METHODS TO DETECT
SPECIFIC NUCLEIC ACID SEQUENCES

THE DEVELOPMENT OF METHODS TO AMPLIFY DNA

THE DEVELOPMENT OF METHODS TO MAKE RECOMBINANT DNA

EVERY MEDICAL STUDENT NEEDS A FUNDAMENTAL UNDERSTANDING OF

THESE MOST IMPORTANT CONCEPTS & METHODS IN MOLECULAR GENETICS
 RESTRICTION ENDONUCLEASES
A RESTRICTION ENDONUCLEASE (RE)
FAITHFULLY BREAKS DNA MOLECULES 5 -G-A-A-T-T-C- 3
AT A SPECIFIC SEQUENCE CALLED A 3 -C-T-T-A-A-G- 5
RESTRICTION SITE

RESTRICTION SITES ARE PALINDROMES = RESTRICTION SITE OF EcoR1: A

SYMMETRICAL INVERTED DNA REPEATS RESTRICTION ENZYME PURIFIED
FROM E. coli
(ARROWS INDICATE BONDS
ACTUALLY HYDROLYZED)
TREATMENT OF DNA HAVING AN EcoR1 HEAT TO
SITE WITH EcoR1 GIVES 2 FRAGMENTS SEPARATE
WITH IDENTICAL 5 OVERLAPPING ENDS STRANDS

5 -G- 3 5 -A-A-T-T-C- 3
MANY DIFFERENT REs HAVE BEEN 3 -C-T-T-A-A- 5 3 -G- 5
PURIFIED FROM BACTERIA
COOL
EACH RE RECOGNIZES A DIFFERENT
RESTRICTION SITE ON DNA COHESIVE ENDS
REANNEAL

5 -G-A-A-T-T-C- 3
3 -C-T-T-A-A-G- 5
 RECOMBINANT DNA
RECOMBINANT DNA CONTAINS TWO DIFFERENT DNA
MOLECULES THAT HAVE BEEN JOINED TOGETHER

FORMATION OF RECOMBINANT DNA

TREATMENT OF 2 DIFFERENT DNA MOLECULES THAT 5 5
HAVE THE SAME RESTRICTION SITE WITH THE
CORRESPONDING RE YIELDS DNA FRAGMENTS THAT
HAVE COMPLEMENTARY OR COHESIVE ENDS

IN THIS EXAMPLE 2 DIFFERENT DNA MOLECULES

EACH WITH AN EcoRI SITE ARE TREATED WITH EcoRI

COMPLEMENTARY DNA FRAGMENTS FROM 5 5

DIFFERENT DNA MOLECULES
HYBRIDIZE & ANNEAL

THE ENZYME DNA LIGASE CLOSES THE

3- 5 PHOSPHODIESTER BONDS TO GIVE
RECOMBINANT DNA MOLECULES
 SOUTHERN BLOTTING
SOUTHERN BLOTTING ALLOWS THE DETECTION
OF A SPECIFIC DNA (OR GENE) SEQUENCE IN A
LARGE SAMPLE OF DNA

DNA IS FIRST CUT BY A RESTRICTION ENDONUCLEASE

(RE) INTO RESTRICTION FRAGMENTS (RFs) OF
VARIABLE LENGTHS
IN THIS EXAMPLE WHOLE GENOMIC DNA ISOLATED FROM 3
DIFFERENT PATIENTS (A,B & C) IS TREATED WITH THE SAME
RE TO PRODUCE THOUSANDS OF DNA FRAGMENTS
EACH PATIENT SAMPLE IS THEN SUBJECTED TO
AGAROSE GEL ELECTROPHORESIS (AGE)
AGE SEPARATES THE RFs ON THE BASIS OF SIZE
At this point
(THE SMALLER FRAGMENTS MOVE FASTER) the DNA is
actually
THE GEL IS OVERLAID WITH A SHEET OF invisible
NITROCELLULOSE FILTER PAPER
OVER THAT IS PLACED SEVERAL LAYERS OF
ABSORBENT BLOTTING PAPER
CAPILLARY ACTION DRAWS THE DNA FRAGMENTS
UP INTO THE NITROCELLULOSE FILTER WHERE
THEY BIND TIGHTLY
 THE BLOTTING PAPER IS PEELED AWAY & THE
NITROCELLULOSE FILTER IS BATHED IN A SOLUTION
CONTAINING A DNA PROBE THAT IS COMPLEMENTARY
TO THE DNA SEQUENCE OF INTEREST
THE DNA PROBE LABELS ONLY FRAGMENTS THAT
CONTAIN PART OF THE DNA SEQUENCE OF INTEREST
THE EXCESS PROBE IS WASHED AWAY & THE
LABELED DNA FRAGMENTS ARE DETECTED At this point
the DNA is still
THE MOST COMMON PROBES USED ARE invisible
32P-LABELED OLIGONUCLEOTIDES WITH DETECTION
Wash away
BY AUTORADIOGRAPHY ON XRAY FILM excess probe

IN THIS EXAMPLE PATIENTS A & B SHOW 3 BANDS

OF VARYING SIZE 1 > 2 > 3 Detect probe
THIS INDICATES THAT THEIR DNA OF INTEREST IS
CUT BY THE RE AT 2 SITES WITHIN ITS SEQUENCE 1 2 3

A
PATIENT 3 SHOWS 4 BANDS INCLUDING 1 & 3
B
BUT BAND 2 HAS BEEN CUT INTO 2 SMALLER
FRAGMENTS (2A & 2B) INDICATING AN ADDITIONAL C

RE SITE IN THE SEQUENCE OF PATIENT 3 2 2

A B
ALTERED DNA FRAGMENTATION IN DIFFERENT
INDIVIDUALS OF THE SAME SPECIES IS CALLED
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
 DNA FINGERPRINTING
DNA FINGERPRINTING IS A METHOD TO
IDENTIFY AN INDIVIDUAL BY UNIQUE
CHARACTERISTICS IN THEIR DNA
DNA FINGERPRINTING HAS BEEN IMPORTANT IN
CRIMINAL INVESTIGATIONS, IN PATERNITY
TESTING & IN IDENTIFYING THE REMAINS OF
UNKNOWN DECEASED PERSONS

DNA FINGERPRINTING WAS ORIGINALLY

PERFORMED USING RFLP ANALYSIS

IN RFLP ANALYSIS AN INDIVIDUAL DNA SAMPLE IS

FRAGMENTED WITH A RESTRICTION ENZYME
FOLLOWED BY SOUTHERN BLOT ANALYSIS TO
PRODUCE A RESTRICTION DIGEST

EACH INDIVIDUAL (EXCEPT FOR MONOZYGOUS

TWINS) DISPLAYS A UNIQUE RESTRICTION DIGEST

AS THE METHOD OF CHOICE FOR DNA

IN THIS DNA FINGERPRINT USED
FINGERPRINTING RFLP ANALYSIS HAS NOW BEEN
AS EVIDENCE IN A MURDER TRIAL
LARGELY REPLACED BY PCR AMPLIFICATION
THE DNA ISOLATED FROM BLOOD
(SEE DNA PROFILING BELOW)
ON THE DEFENDANTS CLOTHING
MATCHES THAT OF THE VICTIM
 NORTHERN BLOTTING
SOUTHERN BLOTTING WAS
DEVELOPED BY E.M. SOUTHERN & IS
NAMED AFTER HIM

SHORTLY AFTER SOUTHERN BLOTTING

WAS DESCRIBED, SEVERAL LABS
DEVELOPED A SIMILAR METHOD TO
DETECT SPECIFIC RNA MOLECULES

SOMEWHAT HUMOROUSLY THEY

NAMED THIS RNA METHOD
NORTHERN BLOTTING
NORTHERN BLOTTING CAN
QUANTIFY THE AMOUNT OF
MESSENGER RNA TRANSCRIBED
FROM A SPECIFIC GENE
NORTHERN BLOTTING HAS BEEN
VALUABLE IN MEASURING THE
EXPRESSION LEVELS OF A SPECIFIC
GENE IN DIFFERENT TISSUES Protein X
mRNA

(FOR AN EXPLANATION OF WESTERN

BLOTTING SEE SIM #1-2 PROTEIN
PURIFICATION & ANALYSIS)
 DNA OF CLONING
BACTERIAL TRANSFORMATION INTEREST VECTOR
ONE METHOD OF AMPLIFYING
RECOMBINANT DNA IS THROUGH
BACTERIAL TRANSFORMATION
THE DNA OF INTEREST IS INSERTED OR
CLONED INTO A CLONING VECTOR USING
RESTRICTION ENDONUCLEASES TO FORM
RECOMBINANT DNA

MOST CLONING VECTORS ARE BACTERIAL

PLASMIDS ENGINEEERED TO CONTAIN
MULTIPLE UNIQUE RESTRICTION SITES

PLASMIDS ARE SMALL CIRCULAR DNA

MOLECULES THAT ARE PRESENT INSIDE
BACTERIA IN MULTIPLE COPIES

THE RESTRICTION SITES IN THE DNA OF IN THIS EXAMPLE THE 2 RESTRICTION

INTEREST MUST ALSO BE KNOWN ENZYMES Sac I & Sal I ARE USED
THEY CAN BE IDENTIFIED BY RESTRICTION
MAPPING OR BY DNA SEQUENCING
ALSO RESTRICTION SITES CAN BE
ENGINEERED INTO THE DNA
RECOMBINANT PLASMIDS ARE MIXED
DNA of
WITH BACTERIA interest

A BRIEF ELEVATION IN TEMPERATURE

Heat
(HEAT SHOCK) RENDERS THE shock
BACTERIAL MEMBRANE PERMEABLE
TO THE PLASMID

THE PLASMID ENTERS SOME OF THE Grow in

antibiotic
BACTERIAL CELLS

THE BACTERIA ARE INCUBATED IN

SELECTIVE MEDIA THAT ALLOWS
ONLY CELLS THAT CONTAIN PLASMIDS
TO MULTIPLY
THIS SELECTION IS USUALLY DONE BY
USING ANTIBIOTIC RESISTANCE
PLASMID VECTORS CONTAIN 1 OR MORE
GENES ENCODING RESISTANCE TO A
SPECIFIC ANTIBIOTIC
ONLY BACTERIA THAT CONTAIN THE
PLASMID WILL GROW IN MEDIA
CONTAINING THE ANTIBIOTIC

ANTIBIOTIC RESISTANCE GENES USUALLY ENCODE

AN ENZYME THAT DEGRADES THE ANTIBIOTIC
 TRANSFORMED BACTERIA ARE
COLLECTED BY CENTRIFUGATION &
LYSED TO RELEASE THEIR CONTENTS

RECOMBINANT PLASMID DNA IS PURIFIED

FROM THE CELL LYSATE USING
ION EXCHANGE CHROMATOGRAPHY

RECOMBINANT PLASMID DNA IS

USUALLY QUANTIFIED USING
ULTRAVIOLET SPECTROPHOTOMETRY
TO MEASURE ABSORBANCE AT 260nm
 EXPRESSION OF EXPRESSION
Hind III EcoR1 VECTOR
FOREIGN GENES
Antibiotic
RECOMBINANT DNA TECHNOLOGY Resistance Promoter
CAN BE USED TO EXPRESS FOREIGN Hind III & EcoR I Gene
GENE PRODUCTS IN BACTERIA
AAGCTT GAATTC
THIS REQUIRES A BACTERIAL TTCGAA CTTAAG
AGCTT G
EXPRESSION VECTOR A CTTA

A BACTERIAL EXPRESSION VECTOR IS

A CLONING VECTOR ENGINEERED TO
CONTAIN THE DNA ELEMENTS NEEDED AGCTT G A AATTC
FOR TRANSCRIPTION & TRANSLATION A CTTAA TTCGA G

THE FOREIGN GENE MUST BE

INSERTED VIA DIRECTIONAL CLONING AAGCTT GAATTC
TO ASSURE IT IS TRANSCRIBED IN THE TTCGAA CTTAAG

CORRECT DIRECTION

FOR DIRECTIONAL CLONING THERE DIRECTIONAL CLONING USING THE

MUST BE 2 DIFFERENT RESTRICTION RESTRICTION ENZYMES EcoRI & Hind III
SITES IN THE SAME ORIENTATION ON
(ARROWS SHOW DIRECTION OF TRANSCRIPTION)
BOTH THE VECTOR & THE DNA
INSERT
 ONE OF THE FIRST APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY WAS TO
MASS PRODUCE HUMAN INSULIN
DIABETIC PATIENTS RELIED ON INSULIN PURIFIED FROM THE PANCREAS OF ANIMALS
BUT ANIMAL-SOURCE INSULIN CAN PRODUCE SEVERE ALLERGIC REACTIONS
POLYMERASE CHAIN REACTION (PCR)
ANOTHER METHOD OF AMPLIFYING
DNA IS THROUGH THE
POLYMERASE CHAIN REACTION (PCR)
Heat to
OLIGONUCLEOTIDE PRIMERS (OLIGOMERS) separate Cool to anneal
COMPLEMENTARY TO VECTOR DNA FLANKING primers
THE DNA INSERT ARE ADDED TO THE
RECOMBINANT PLASMID

A HEAT-STABLE DNA POLYMERASE & FREE

DEOXYNUCLEOTIDES (dNTPs) ARE ALSO ADDED

THE MIXTURE IS HEATED TO ABOUT 90oC Heat-Stable

TO SEPARATE THE DNA STRANDS Heat/Cool
+
NTPs
THEN THE MIXTURE IS COOLED
TO ANNEAL THE PRIMERS

TIME IS ALLOWED FOR THE DNA POLYMERASE

TO EXTEND THE NEW STRANDS
 THE NEW DNA IS THEN HEATED AGAIN TO
SEPARATE THE STRANDS & COOLED AGAIN TO
ALLOW NEW OLIGOMERS TO ANNEAL

THIS PROCESS IS REPEATED FOR SEVERAL CYCLES

Heat to
THE DNA OF INTEREST IS AMPLIFIED EXPONENTIALLY separate Cool to anneal
primers
THEORETICALLY: n CYCLES GENERATE
2n COPIES FROM 1 PLASMID
FOR EXAMPLE 20 CYCLES CAN GENERATE
220 OR 1,048,576 COPIES FROM 1 PLASMID

USING HEAT-STABLE DNA POLYMERASE Heat-Stable

ELIMINATES THE NEED TO ADD NEW Heat/Cool
+
POLYMERASE AFTER EACH HEATING NTPs

HEAT-STABLE DNA POLYMERASE ALLOWS FOR

AUTOMATIC PCR CYCLING

THE FIRST HEAT-STABLE DNA POLYMERASE

WASTaq POLYMERASE ISOLATED FROM Thermus
aquaticus BACTERIA LIVING IN THE HOT SPRINGS
OF YELLOWSTONE NATIONAL PARK
PCR CAN AMPLIFY A DNA SEGMENT DIRECTLY DNA OF
FROM AN ORGANISMS WHOLE GENOMIC DNA !! INTEREST
(IF THE SEQUENCE OF THE DNA SEGMENT
IS ALREADY KNOWN)

THE USE OF GENE-SPECIFIC PRIMERS LIMITS

THE AMPLIFICATION TO THE SEGMENT
WHOLE
GENOMIC DNA
THE REST OF THE GENOMIC DNA IS ADD
NOT AMPLIFIED GENE-SPECIFIC
PRIMERS
THE AMPLIFIED SEGMENT CAN BE EASILY
SEPARATED FROM THE GENOMIC DNA
DUE TO ITS SMALLER LENGTH

PCR
 DNA PROFILING
DNA PROFILING IS A METHOD TO IDENTIFY AN
INDIVIDUAL BY THE UNIQUENESS OF THEIR DNA
DNA PROFILING IS VERY IMPORTANT IN PATERNITY
TESTING, IN CRIMINAL INVESTIGATIONS & IN
IDENTIFYING DECEASED REMAINS

DNA PROFILING WAS ORIGINALLY CALLED

DNA FINGERPRINTING (SEE ABOVE)

DNA FINGERPRINTING WAS ORIGINALLY

PERFORMED USING RFLP ANALYSIS

BUT RFLP ANALYSIS IS SLOW & REQUIRES

RELATIVELY LARGE AMOUNTS OF DNA

DNA PROFILING IS NOW DONE BY PCR PCR THERMAL

AMPLIFICATION FOLLOWED BY SEPARATION & CYCLER
DETECTION ON ELECTROPHORESIS &
QUANTIFICATION ON SPECTROPHOTOMETRY

ONLY TINY AMOUNTS OF DNA ARE NEEDED & THE

METHOD IS MUCH FASTER THAN RFLP ANALYSIS
IN DNA PROFILING THE PCR PRIMERS AMPLIFY
VARIABLE NUMBER TANDEM REPEATS (VNTRs)

VNTRs ARE NON-CODING NUCLEOTIDE SEQUENCES

ARRANGED IN TANDEM REPEATS THAT DISPLAY
HIGH VARIABILITY AMONG INDIVIDUALS OF THE
SAME SPECIES (EXCEPT FOR MONZYGOTIC TWINS)

VNTR VARIANTS SHOW MENDELIAN INHERITANCE ELECTROPHORESIS OF PCR-

DNA PROFILING CURRENTLY USES A SET OF AMPLIFIED DNA USING PRIMERS
VERY SHORT VNTRs OF 3 5 BASE PAIRS FOR 12 DIFFERENT STRs
CALLED SHORT-TERM REPEATS (STRs) (DETECTION BY ETHIDIUM BROMIDE)
THE PROBABILITY THAT 2 UNRELATED
INDIVIDUALS SHARE 1 STR ALLELE IS ABOUT 1/10
THE PROBABILITY THAT 2 UNRELATED
INDIVIDUALS SHARE 10 STR ALLELES IS ABOUT
1/10,000,000,000

THE COMBINED DNA INDEX SYSTEM (CODIS) DNA PROFILE PRINTOUT READ
MAINTAINED BY THE FBI UTILIZES 13 STR LOCI FROM PCR/ELECTROPHORESIS
ON 13 DIFFERENT HUMAN CHROMOSOMES USING 10 DIFFERENT STRs
 DNA SEQUENCING
THE DIRECT SEQUENCING OF DNA HAS BEEN A
MAJOR ADVANCEMENT IN MOLECULAR GENETICS

THE CURRENT DNA SEQUENCING METHOD OF dNTP

CHOICE IS THE INTERRUPTED ENZYMATIC-
CLEAVAGE METHOD OR THE SANGER METHOD

THE SANGER METHOD DEPENDS ON THE USE OF

DIDEOXYNUCLEOSIDE TRIPHOSPHATES (ddNTPs)

ddNTPs ARE INCORPORATED INTO A DNA CHAIN ddNTP

BY DNA POLYMERASES JUST LIKE dNTPs

BUT ddNTPs LACK THE 3 OH GROUP THAT IS

PRESENT ON dNTPs

SO INCORPORATION OF A ddNTP INTO A CHAIN

INTERRUPTS (STOPS) ELONGATION OF THAT CHAIN
 AMPLIFIED DNA OF INTEREST IS MIXED WITH A
LABELED PRIMER THAT IS 5 3 COMPLEMENTARY
TO THE VECTOR DNA AT THE 3 END OF THE
ANTISENSE STRAND
THE PRIMER MUST BE LABELED WITH A
RADIOISOTOPE (AS SHOWN HERE) OR
WITH A CHEMICAL LABEL

THE MIXTURE IS HEATED TO SEPARATE THE

STRANDS THEN COOLED TO ANNEAL THE PRIMER
THE MIXTURE IS DIVIDED INTO 4 SAMPLES

EACH SAMPLE CONTAINS A DNA POLYMERASE

ALONG WITH THE 4 dNTPs &
ONLY ONE OF THE 4 ddNTPs
(ONLY THE SAMPLE WITH ddGTP IS SHOWN HERE)

INTERRUPTION OF DNA SYNTHESIS OCCURS

RANDOMLY AT ALL POSSIBLE COMPLEMENTARY
SITES ON THE DNA TMEPLATE (IN THIS CASE C)

THIS GIVES A MIXTURE OF LABELED DNA

MOLECULES OF DIFFERENT LENGTHS
 THE 4 SAMPLES ARE SUBJECTED TO
POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

PAGE SEPARATES THE CHAINS ON THE BASIS

OF SIZE (LENGTH) 5
THE SMALLEST CHAINS MOVE THE FASTEST

THE LABELED SPECIES ARE DETECTED

(IN THIS CASE BY AUTORADIOGRAPHY)

THE 5 3 SEQUENCE OF THE SENSE STRAND

IS READ FROM SMALLEST TO LARGEST

(NOTE THAT THIS PAGE DOES NOT

CORRESPOND TO THE SAMPLE
SHOWN ON THE PREVIOUS SLIDE)

(FOR AN EXPLANATION OF PAGE SEE SIM #1-2

PROTEIN PURIFICATION & ANALYSIS)
DNA SEQUENCING IS NOW
COMPLETELY AUTOMATED

A DNA
SEQUENCER

BECKMAN-COULTER
DNA SEQUENCER
PRINT-OUT FROM A DNA SEQUENCER
RED = T
GREEN = C
BLACK = A
YELLOW = G
 CLINICAL CORRELATION O2 SATURATION CURVE
Normal
CHARACTERIZING THE MOLECULAR BASIS
OF A GENETIC DISEASE
Patient
CASE REPORT
A 3 year-old boy was suspected of having a
hemoglobinopathy. His parents said that since birth he
occasionally turned blue. After activity he often
squatted as if he were trying to catch his breath. He
was not very active & he slept more than is normal.
His pediatricians had ruled out cardiovascular &
pulmonary causes of his cyanosis.

An oxygen saturation curve on the patients

pH 8.5
erythrocytes showed a shift to the right indicating
decreased affinity for oxygen. O

Hemoglobin electrophoresis was inconclusive because

it showed a single major band with the same mobility
as normal hemoglobin (HbA1).

Molecular genetics tests were ordered.

+
HbA1 Pt HbS

NON-DENATURING
ELECTROPHORESIS
(HbS IS SICKLE CELL
HEMOGLOBIN)
Patient DNA was isolated from a blood sample.
3 separate PCR amplifications were performed Patient Patient
using the DNA. Each PCR used oligonucleotide Blood DNA
primers (green arrows) for DNA flanking one of
the 3 exons of the beta-globin gene. The
amplified DNA was isolated & sequenced.

DNA sequencing revealed an abnormal base Exon 1 Exon 2 Exon 3

substitution in the DNA encoding the second
exon. This point mutation (AAC AGC) at -Globin gene on chromosome 11
position 2 in codon 102 predicted an amino
acid substitution of serine in place of normal
asparagine at that position. Exon 2 with normal PCR 1 PCR 3
PCR 2
sequence was also observed.
Exon 1 Exon 2 Exon 3
The patient was diagnosed as being
heterozygous for Hemoglobin Beth Israel which ISOL ATE
was first described in 1976.
SEQUEN CE DNA
Hb BETH ISRAEL: 2(Asn102Ser)
NORMAL 102AAC AGC NORMAL
-Asparagine 102 forms a hydrogen bond with SEQUENCE SEQUENCE
-aspartate 94 in the oxy (R) conformation of
hemoglobin & stabilizes the oxy conformation. NORMAL
-Serine 102 cannot form a hydrogen bond with SEQUENCE
-aspartate 94. This destabilizes the oxy
conformation resulting in lower O2 affinity.
THE COMBINATION OF PCR AMPLIFICATION & DIRECT
DNA SEQUENCING WAS USED TO SEQUENCE THE
HUMAN GENOME IN 2000

AS A RESULT OF THE SEQUENCING OF THE

HUMAN GENOME THE SEQUENCE OF ALL
HUMAN GENES IS NOW KNOWN
BASED ON THIS INFORMATION PCR PRIMERS &
DNA SEQUENCING PRIMERS CAN BE DESIGNED
FOR THE PROTEIN-ENCODING EXONS OF EVERY
HUMAN GENE AS WELL AS FOR UNTRANSLATED
HUMAN DNA SEQUENCES INVOLVED IN DISEASE
J. CRAIG VENTER &
THE COMBINATION OF PCR AMPLIFICATION & DIRECT FRANCIS COLLINS ON
DNA SEQUENCING HAS BECOME THE METHOD OF THE JUNE 4, 2000
CHOICE TO IDENTIFY THE DNA MUTATIONS THAT CAUSE COVER OF TIME
SPECIFIC HUMAN GENETIC DISEASES

THIS STRATEGY HAS ALSO NOW MADE IT

POSSIBLE TO DIAGNOSE SOME GENETIC
DISEASES IN INDIVIDUAL PATIENTS
END OF SIM 1-5